RNA interference mediated inhibition of myostatin gene expression using short interfering nucleic acid (siNA)

ABSTRACT

This invention relates to compounds, compositions, and methods useful for modulating myostatin (GDF8) gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of myostatin gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of myostatin genes.

This application is a continuation of U.S. patent application Ser. No.12,185,678 filed on Aug. 4, 2008, now abandoned which is a continuationof U.S. patent application Ser. No. 10/879,867, filed on Jun. 28, 2004(now abandoned), which is a continuation-in-part of International PatentApplication No. PCT/US04/16390, filed May 24, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/826,966,filed Apr. 16, 2004 (now abandoned), which is continuation-in-part ofU.S. patent application Ser. No. 10/757,803, filed Jan. 14, 2004, whichis a continuation-in-part of U.S. patent application Ser. No.10/720,448, filed Nov. 24, 2003, which is a continuation-in-part of U.S.patent application Ser. No. 10/693,059, filed Oct. 23, 2003, nowabandoned which is a continuation-in-part of U.S. patent applicationSer. No. 10/444,853, filed May 23, 2003, which is a continuation-in-partof International Patent Application No. PCT/US03/05346, filed Feb. 20,2003, and a continuation-in-part of International Patent Application No.PCT/US03/05028, filed Feb. 20, 2003, both of which claim the benefit ofU.S. Provisional Application No. 60/358,580 filed Feb. 20, 2002 (nowexpired), U.S. Provisional Application No. 60/363,124 filed Mar. 11,2002 (now expired), U.S. Provisional Application No. 60/386,782 filedJun. 6, 2002 (now expired), U.S. Provisional Application No. 60/406,784filed Aug. 29, 2002 (now expired), U.S. Provisional Application No.60/408,378 filed Sep. 5, 2002 (now expired), U.S. ProvisionalApplication No. 60/409,293 filed Sep. 9, 2002 (now expired), and U.S.Provisional Application No. 60/440,129 filed Jan. 15, 2003 (nowexpired). The instant application claims the benefit of all the listedapplications, which are hereby incorporated by reference herein in theirentireties, including the drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SubstituteSQL58USCNT2,”created on May 19, 2010, which is 150,550 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of myostatin expression and/oractivity. The present invention is also directed to compounds,compositions, and methods relating to traits, diseases and conditionsthat respond to the modulation of expression and/or activity of genesinvolved in myostatin gene expression pathways or other cellularprocesses that mediate the maintenance or development of such traits,diseases and conditions. Specifically, the invention relates to smallnucleic acid molecules, such as short interfering nucleic acid (siNA),short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA(miRNA), and short hairpin RNA (shRNA) molecules capable of mediatingRNA interference (RNAi) against myostatin gene expression. Such smallnucleic acid molecules are useful, for example, in providingcompositions for the treatment or prevention of diseases and conditionsassociated with muscle atrophy, weakness and/or degeneration, such asmuscular dystrophy, myotonic dystrophy, muscle wasting, sarcopenia,myalgias, myopathies, hypotonis, cachexia, spinal cord injury, or muscleinjury, for treating or preventing obesity, diabetes (e.g., type I andtype II), and insulin resistance, or alternately for providingcompositions for muscle hypertrophy, including use for increasedstrength, athleticism, bodybuilding, or cosmetic applications.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of an siRNAduplex is required for siRNA activity and that ATP is utilized tomaintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation of doublestranded RNA-dependent protein kinase PKR, specifically 2′-amino or2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certain singlestranded siRNA constructs, including certain 5′-phosphorylated singlestranded siRNAs that mediate RNA interference in HeLa cells. Harborth etal., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105,describe certain chemically and structurally modified siRNA molecules.Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically andstructurally modified siRNA molecules. Woolf et al., International PCTPublication Nos. WO 03/064626 and WO 03/064625 describe certainchemically modified dsRNA constructs.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating myostatin gene expression using short interfering nucleicacid (siNA) molecules. This invention also relates to compounds,compositions, and methods useful for modulating the expression andactivity of other genes involved in pathways of myostatin geneexpression and/or activity by RNA interference (RNAi) using smallnucleic acid molecules. In particular, the instant invention featuressmall nucleic acid molecules, such as short interfering nucleic acid(siNA), short interfering RNA (siRNA), double stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methodsused to modulate the expression of myostatin genes.

An siNA of the invention can be unmodified or chemically modified. AnsiNA of the instant invention can be chemically synthesized, expressedfrom a vector or enzymatically synthesized. The instant invention alsofeatures various chemically modified synthetic short interfering nucleicacid (siNA) molecules capable of modulating myostatin gene expression oractivity in cells by RNA interference (RNAi). The use of chemicallymodified siNA improves various properties of native siNA moleculesthrough increased resistance to nuclease degradation in vivo and/orthrough improved cellular uptake. Further, contrary to earlier publishedstudies, siNA having multiple chemical modifications retains its RNAiactivity. The siNA molecules of the instant invention provide usefulreagents and methods for a variety of therapeutic, diagnostic, targetvalidation, genomic discovery, genetic engineering, and pharmacogenomicapplications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofmyostatin genes encoding proteins, such as proteins comprisingmyostatin, such as genes encoding sequences comprising those sequencesreferred to by GenBank Accession Nos. shown in Table I, referred toherein generally as myostatin but also known as MSTN or GrowthDifferentiation Factor-8 (GDF-8). The description below of the variousaspects and embodiments of the invention is provided with reference toexemplary myostatin/MSTN/GDF-8 gene referred to herein as myostatin.However, the various aspects and embodiments are also directed to othermyostatin genes, such as myostatin homolog genes, transcript variants,and polymorphisms (e.g., single nucleotide polymorphism, (SNPs))associated with certain myostatin genes, for example genes associatedwith diseases, traits, or conditions described herein or otherwise knownin the art. As such, the various aspects and embodiments are alsodirected to other genes that are involved in myostatin mediated pathwaysof signal transduction or gene expression. These additional genes can beanalyzed for target sites using the methods described for myostatingenes herein. Thus, the modulation of other genes and the effects ofsuch modulation of the other genes can be performed, determined, andmeasured as described herein.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a myostatin gene, wherein said siNA molecule comprises about 18 toabout 21 base pairs.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of amyostatin RNA via RNA interference (RNAi), wherein the double strandedsiNA molecule comprises a first and a second strand, each strand of thesiNA molecule is about 18 to about 23 nucleotides in length, the firststrand of the siNA molecule comprises nucleotide sequence havingsufficient complementarity to the myostatin RNA for the siNA molecule todirect cleavage of the myostatin RNA via RNA interference, and thesecond strand of said siNA molecule comprises nucleotide sequence thatis complementary to the first strand.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a myostatin RNA via RNA interference (RNAi), whereineach strand of the siNA molecule is about 18 to about 23 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the myostatin RNA for thesiNA molecule to direct cleavage of the myostatin RNA via RNAinterference.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a myostatin gene, for example, wherein themyostatin gene comprises myostatin encoding sequence. In one embodiment,the invention features an siNA molecule that down-regulates expressionof a myostatin gene, for example, wherein the myostatin gene comprisesmyostatin non-coding sequence or regulatory elements involved inmyostatin gene expression.

In one embodiment, an siNA of the invention is used to inhibit theexpression of a myostatin gene or a myostatin gene family, wherein thegenes or gene family sequences share sequence homology. Such homologoussequences can be identified as is known in the art, for example usingsequence alignments. siNA molecules can be designed to target suchhomologous sequences, for example using perfectly complementarysequences or by incorporating non-canonical base pairs, for examplemismatches and/or wobble base pairs that can provide additional targetsequences. In instances where mismatches are identified, non-canonicalbase pairs (for example, mismatches and/or wobble bases) can be used togenerate siNA molecules that target more than one gene sequence. In anon-limiting example, non-canonical base pairs such as UU and CC basepairs are used to generate siNA molecules that are capable of targetingsequences for differing myostatin targets that share sequence homology(e.g., other myostatin encoding sequences). As such, one advantage ofusing siNAs of the invention is that a single siNA can be designed toinclude nucleic acid sequence that is complementary to the nucleotidesequence that is conserved between the homologous genes. In thisapproach, a single siNA can be used to inhibit expression of more thanone gene instead of using more than one siNA molecule to target thedifferent genes.

In one embodiment, the invention features an siNA molecule having RNAiactivity against myostatin RNA, wherein the siNA molecule comprises asequence complementary to any RNA having myostatin encoding sequence,such as those sequences having GenBank Accession Nos. shown in Table I.In another embodiment, the invention features an siNA molecule havingRNAi activity against myostatin RNA, wherein the siNA molecule comprisesa sequence complementary to an RNA having variant myostatin encodingsequence, for example other mutant myostatin genes not shown in Table Ibut known in the art to be associated with neuronal apoptosis. Chemicalmodifications as shown in Tables III and IV or otherwise describedherein can be applied to any siNA construct of the invention. In anotherembodiment, an siNA molecule of the invention includes a nucleotidesequence that can interact with nucleotide sequence of a myostatin geneand thereby mediate silencing of myostatin gene expression, for example,wherein the siNA mediates regulation of myostatin gene expression bycellular processes that modulate the chromatin structure or methylationpatterns of the myostatin gene and prevent transcription of themyostatin gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of myostatin proteins arising frommyostatin haplotype polymorphisms that are associated with a disease orcondition, (e.g., muscle catabolism, muscle atrophy, muscle weaknessetc.). Analysis of myostatin genes, or myostatin protein or RNA levelscan be used to identify subjects with such polymorphisms or thosesubjects who are at risk of developing traits, conditions, or diseasesdescribed herein. These subjects are amenable to treatment, for example,treatment with siNA molecules of the invention and any other compositionuseful in treating diseases or conditions related to myostatin geneexpression. As such, analysis of myostatin protein or RNA levels can beused to determine treatment type and the course of therapy in treating asubject. Monitoring of myostatin protein or RNA levels can be used topredict treatment outcome and to determine the efficacy of compounds andcompositions that modulate the level and/or activity of certainmyostatin proteins associated with a trait, condition, or disease.

In one embodiment of the invention an siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a myostatinprotein. The siNA further comprises a sense strand, wherein said sensestrand comprises a nucleotide sequence of a myostatin gene or a portionthereof.

In another embodiment, an siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a myostatin protein or a portion thereof. The siNAmolecule further comprises a sense region, wherein said sense regioncomprises a nucleotide sequence of a myostatin gene or a portionthereof.

In another embodiment, the invention features an siNA moleculecomprising a nucleotide sequence in the antisense region of the siNAmolecule that is complementary to a nucleotide sequence or portion ofsequence of a myostatin gene. In another embodiment, the inventionfeatures an siNA molecule comprising a region, for example, theantisense region of the siNA construct, complementary to a sequencecomprising a myostatin gene sequence or a portion thereof.

In one embodiment, the antisense region of myostatin siNA constructscomprises a sequence complementary to sequence having any of SEQ ID NOs.1-157 or 315-322. In one embodiment, the antisense region of myostatinconstructs comprises sequence having any of SEQ ID NOs. 158-314,331-338, 347-354, 363-370, 379-386, 395-418, 420, 422, 424, 427, 429,431, 433, or 436. In another embodiment, the sense region of myostatinconstructs comprises sequence having any of SEQ ID NOs. 1-157, 315-330,339-346, 355-362, 371-378, 387-394, 419, 421, 423, 425, 426, 428, 430,432, 434, or 435.

In one embodiment, an siNA molecule of the invention can comprise any ofSEQ ID Nos 1-436. The sequences shown in SEQ ID NOs: 1-436 are notlimiting. An siNA molecule of the invention can comprise any contiguousmyostatin sequence (e.g., about 18 to about 25, or about 18, 19, 20, 21,22, 23, 24, or 25 contiguous myostatin nucleotides).

In yet another embodiment, the invention features an siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siNA construct of the invention.

In one embodiment of the invention an siNA molecule comprises anantisense strand having about 19 to about 29 (e.g., about 19, 20, 21,22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein the antisensestrand is complementary to a RNA sequence encoding a myostatin protein,and wherein said siNA further comprises a sense strand having about 19to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29)nucleotides, and wherein said sense strand and said antisense strand aredistinct nucleotide sequences with at least about 19 complementarynucleotides.

In another embodiment of the invention an siNA molecule of the inventioncomprises an antisense region having about 19 to about 29 (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein theantisense region is complementary to a RNA sequence encoding a myostatinprotein, and wherein said siNA further comprises a sense region havingabout 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27,28, or 29) nucleotides, wherein said sense region and said antisenseregion comprise a linear molecule with at least about 19 complementarynucleotides.

In one embodiment, an siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a myostatin gene. Becausemyostatin genes can share some degree of sequence homology with eachother, siNA molecules can be designed to target a class of myostatingenes or alternately specific myostatin genes (e.g., myostatinpolymorphic variants) by selecting sequences that are either sharedamongst different myostatin targets or alternatively that are unique fora specific myostatin target (e.g., DNA or RNA encoding myostatin).Therefore, in one embodiment, the siNA molecule can be designed totarget conserved regions of myostatin RNA sequences having homologyamong several myostatin gene variants so as to target a class ofmyostatin genes with one siNA molecule. Accordingly, in one embodiment,the siNA molecule of the invention modulates the expression of one orboth myostatin alleles in a subject. In another embodiment, the siNAmolecule can be designed to target a sequence that is unique to aspecific myostatin RNA sequence (e.g., a single myostatin allele ormyostatin single nucleotide polymorphism (SNP)) due to the high degreeof specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response are doublestranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplex nucleic acid moleculescontaining about 19 base pairs between oligonucleotides comprising about19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides.In yet another embodiment, siNA molecules of the invention compriseduplex nucleic acid molecules with overhanging ends of about 1 to about3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21-nucleotideduplexes with about 19 base pairs and 3′-terminal mononucleotide,dinucleotide, or trinucleotide overhangs.

In one embodiment, the invention features one or more chemicallymodified siNA constructs having specificity for myostatin expressingnucleic acid molecules, such as RNA encoding a myostatin protein. In oneembodiment, the invention features a RNA based siNA molecule (e.g., ansiNA comprising 2′-OH nucleotides) having specificity for myostatinexpressing nucleic acid molecules that includes one or more chemicalmodifications described herein. Non-limiting examples of such chemicalmodifications include without limitation phosphorothioateinternucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminalglyceryl and/or inverted deoxy abasic residue incorporation. Thesechemical modifications, when used in various siNA constructs (e.g., RNAbased siNA constructs), are shown to preserve RNAi activity in cellswhile at the same time, dramatically increasing the serum stability ofthese compounds. Furthermore, contrary to the data published by Parrishet al., supra, applicant demonstrates that multiple (greater than one)phosphorothioate substitutions are well-tolerated and confer substantialincreases in serum stability for modified siNA constructs.

In one embodiment, an siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, ansiNA molecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, an siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single stranded, the percent modification can be based uponthe total number of nucleotides present in the single stranded siNAmolecules Likewise, if the siNA molecule is double stranded, the percentmodification can be based upon the total number of nucleotides presentin the sense strand, antisense strand, or both the sense and antisensestrands.

One aspect of the invention features a double stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of amyostatin gene. In one embodiment, the double stranded siNA moleculecomprises one or more chemical modifications and each strand of thedouble stranded siNA is about 21 nucleotides long. In one embodiment,the double stranded siNA molecule does not contain any ribonucleotides.In another embodiment, the double stranded siNA molecule comprises oneor more ribonucleotides. In one embodiment, each strand of the doublestranded siNA molecule comprises about 19 to about 29 (e.g., about 19,20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein eachstrand comprises about 19 nucleotides that are complementary to thenucleotides of the other strand. In one embodiment, one of the strandsof the double stranded siNA molecule comprises a nucleotide sequencethat is complementary to a nucleotide sequence or a portion thereof ofthe myostatin gene, and the second strand of the double stranded siNAmolecule comprises a nucleotide sequence substantially similar to thenucleotide sequence of the myostatin gene or a portion thereof.

In another embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a myostatin gene comprising an antisense region, wherein theantisense region comprises a nucleotide sequence that is complementaryto a nucleotide sequence of the myostatin gene or a portion thereof, anda sense region, wherein the sense region comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the myostatin geneor a portion thereof. In one embodiment, the antisense region and thesense region each comprise about 18 to about 23 (e.g. about 18, 19, 20,21, 22, or 23) nucleotides, wherein the antisense region comprises about18 nucleotides that are complementary to nucleotides of the senseregion.

In another embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a myostatin gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the myostatingene or a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region.

In one embodiment, an siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, an siNA molecule comprising modifications described herein(e.g., comprising nucleotides having Formulae I-VII or siNA constructscomprising “Stab 00”-“Stab 25” (Table IV) or any combination thereof(see Table IV)) and/or any length described herein can comprise bluntends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, an siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 18 to about 30nucleotides (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleotides). Other nucleotides present in a blunt ended siNAmolecule can comprise, for example, mismatches, bulges, loops, or wobblebase pairs to modulate the activity of the siNA molecule to mediate RNAinterference.

By “blunt ends” is meant symmetric termini or termini of a doublestranded siNA molecule having no overhanging nucleotides. The twostrands of a double stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a myostatin gene, wherein the siNA molecule is assembled from twoseparate oligonucleotide fragments wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. The sense region can be connected to the antisenseregion via a linker molecule, such as a polynucleotide linker or anon-nucleotide linker.

In one embodiment, the invention features double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a myostatin gene, wherein the siNA molecule comprises about 18 toabout 23 base pairs, and wherein each strand of the siNA moleculecomprises one or more chemical modifications. In another embodiment, oneof the strands of the double stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of amyostatin gene or a portion thereof, and the second strand of the doublestranded siNA molecule comprises a nucleotide sequence substantiallysimilar to the nucleotide sequence or a portion thereof of the myostatingene. In another embodiment, one of the strands of the double strandedsiNA molecule comprises a nucleotide sequence that is complementary to anucleotide sequence of a myostatin gene or portion thereof, and thesecond strand of the double stranded siNA molecule comprises anucleotide sequence substantially similar to the nucleotide sequence orportion thereof of the myostatin gene. In another embodiment, eachstrand of the siNA molecule comprises about 18 to about 23 nucleotides,and each strand comprises at least about 18 nucleotides that arecomplementary to the nucleotides of the other strand. The myostatin genecan comprise, for example, sequences referred to in Table I.

In one embodiment, an siNA molecule of the invention comprises noribonucleotides. In another embodiment, an siNA molecule of theinvention comprises ribonucleotides.

In one embodiment, an siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a myostatin gene or a portion thereof, andthe siNA further comprises a sense region comprising a nucleotidesequence substantially similar to the nucleotide sequence of themyostatin gene or a portion thereof. In another embodiment, theantisense region and the sense region each comprise about 18 to about 23nucleotides and the antisense region comprises at least about 18nucleotides that are complementary to nucleotides of the sense region.The myostatin gene can comprise, for example, sequences referred to inTable I.

In one embodiment, an siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a myostatin gene, or a portion thereof, and the senseregion comprises a nucleotide sequence that is complementary to theantisense region. In one embodiment, the siNA molecule is assembled fromtwo separate oligonucleotide fragments, wherein one fragment comprisesthe sense region and the second fragment comprises the antisense regionof the siNA molecule. In another embodiment, the sense region isconnected to the antisense region via a linker molecule. In anotherembodiment, the sense region is connected to the antisense region via alinker molecule, such as a nucleotide or non-nucleotide linker. Themyostatin gene can comprise, for example, sequences referred in to TableI.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a myostatin gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the myostatingene or a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region, and wherein thesiNA molecule has one or more modified pyrimidine and/or purinenucleotides. In one embodiment, the pyrimidine nucleotides in the senseregion are 2′-O-methyl pyrimidine nucleotides or 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-O-methyl purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In one embodiment, thepyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in theantisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. Inanother embodiment of any of the above-described siNA molecules, anynucleotides present in a non-complementary region of the sense strand(e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a myostatin gene, wherein the siNA molecule is assembled from twoseparate oligonucleotide fragments wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule, and wherein the fragment comprising the sense regionincludes a terminal cap moiety at the 5′-end, the 3′-end, or both of the5′ and 3′ ends of the fragment. In one embodiment, the terminal capmoiety is an inverted deoxy abasic moiety or glyceryl moiety. In oneembodiment, each of the two fragments of the siNA molecule compriseabout 21 nucleotides.

In one embodiment, the invention features an siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, of lengthbetween about 12 and about 36 nucleotides. In one embodiment, allpyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides. In one embodiment, the modified nucleotides inthe siNA include at least one 2′-deoxy-2′-fluoro cytidine or2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, themodified nucleotides in the siNA include at least one 2′-deoxy-2′-fluorocytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In oneembodiment, all uridine nucleotides present in the siNA are2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment,all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified internucleotidic linkage, such as phosphorothioate linkage. Inone embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a method of increasing thestability of an siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment,the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-deoxy-2′-fluoro cytidine and at least one2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridinenucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a myostatin gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the myostatingene or a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region, and wherein thepurine nucleotides present in the antisense region comprise2′-deoxy-purine nucleotides. In an alternative embodiment, the purinenucleotides present in the antisense region comprise 2′-O-methyl purinenucleotides. In either of the above embodiments, the antisense regioncan comprise a phosphorothioate internucleotide linkage at the 3′ end ofthe antisense region. Alternatively, in either of the above embodiments,the antisense region can comprise a glyceryl modification at the 3′ endof the antisense region. In another embodiment of any of theabove-described siNA molecules, any nucleotides present in anon-complementary region of the antisense strand (e.g. overhang region)are 2′-deoxy nucleotides.

In one embodiment, the antisense region of an siNA molecule of theinvention comprises sequence complementary to a portion of a myostatintranscript having sequence unique to a particular myostatin disease,condition, or trait related allele, such as sequence comprising a singlenucleotide polymorphism (SNP) associated with the disease specificdisease, condition, or trait. As such, the antisense region of an siNAmolecule of the invention can comprise sequence complementary tosequences that are unique to a particular allele to provide specificityin mediating selective RNAi against the disease, condition, or traitrelated allele.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a myostatin gene, wherein the siNA molecule is assembled from twoseparate oligonucleotide fragments wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment about 19 nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule and wherein atleast two 3′ terminal nucleotides of each fragment of the siNA moleculeare not base-paired to the nucleotides of the other fragment of the siNAmolecule. In one embodiment, each of the two 3′ terminal nucleotides ofeach fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide,such as a 2′-deoxy-thymidine. In another embodiment, all 21 nucleotidesof each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule. Inanother embodiment, about 19 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the myostatin gene. In another embodiment, about 21nucleotides of the antisense region are base-paired to the nucleotidesequence or a portion thereof of the RNA encoded by the myostatin gene.In any of the above embodiments, the 5′-end of the fragment comprisingsaid antisense region can optionally includes a phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa myostatin RNA sequence (e.g., wherein said target RNA sequence isencoded by a myostatin gene involved in the myostatin pathway), whereinthe siNA molecule does not contain any ribonucleotides and wherein eachstrand of the double stranded siNA molecule is about 18 to about 23nucleotides long. Examples of non-ribonucleotide containing siNAconstructs are combinations of stabilization chemistries shown in TableIV in any combination of Sense/Antisense chemistries, such as Stab 7/8,Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, or Stab18/20.

In one embodiment, the invention features a chemically synthesizeddouble stranded RNA molecule that directs cleavage of a myostatin RNAvia RNA interference, wherein each strand of said RNA molecule is about18 to about 23 nucleotides in length; one strand of the RNA moleculecomprises nucleotide sequence having sufficient complementarity to themyostatin RNA for the RNA molecule to direct cleavage of the myostatinRNA via RNA interference; and wherein at least one strand of the RNAmolecule comprises one or more chemically modified nucleotides describedherein, such as deoxynucleotides, 2′-O-methyl nucleotides,2′-deoxy-2′-fluoro nucleotides, 2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising ansiNA molecule of the invention.

In one embodiment, the invention features an active ingredientcomprising an siNA molecule of the invention.

In one embodiment, the invention features the use of a double strandedshort interfering nucleic acid (siNA) molecule to inhibit,down-regulate, or reduce expression of a myostatin gene, wherein thesiNA molecule comprises one or more chemical modifications and eachstrand of the double stranded siNA is about 18 to about 28 or more(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 or more)nucleotides long.

In one embodiment, the invention features the use of a double strandedshort interfering nucleic acid (siNA) molecule that inhibits,down-regulates, or reduces expression of a myostatin gene, wherein oneof the strands of the double stranded siNA molecule is an antisensestrand which comprises nucleotide sequence that is complementary tonucleotide sequence of myostatin RNA or a portion thereof, the otherstrand is a sense strand which comprises nucleotide sequence that iscomplementary to a nucleotide sequence of the antisense strand andwherein a majority of the pyrimidine nucleotides present in the doublestranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a myostatin gene, wherein one of the strands ofthe double stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence ofmyostatin RNA or a portion thereof, wherein the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a myostatin gene, wherein one of the strands ofthe double stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence ofmyostatin RNA that encodes a protein or portion thereof, the otherstrand is a sense strand which comprises nucleotide sequence that iscomplementary to a nucleotide sequence of the antisense strand andwherein a majority of the pyrimidine nucleotides present in the doublestranded siNA molecule comprises a sugar modification. In oneembodiment, each strand of the siNA molecule comprises about 18 to about29 or more (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or29 or more) nucleotides, wherein each strand comprises at least about 18nucleotides that are complementary to the nucleotides of the otherstrand. In one embodiment, the siNA molecule is assembled from twooligonucleotide fragments, wherein one fragment comprises the nucleotidesequence of the antisense strand of the siNA molecule and a secondfragment comprises nucleotide sequence of the sense region of the siNAmolecule. In one embodiment, the sense strand is connected to theantisense strand via a linker molecule, such as a polynucleotide linkeror a non-nucleotide linker. In a further embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In still another embodiment, thepyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-deoxy purine nucleotides. Inanother embodiment, the antisense strand comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methylpurine nucleotides. In another embodiment, the pyrimidine nucleotidespresent in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and any purine nucleotides present in the antisense strandare 2′-O-methyl purine nucleotides. In a further embodiment the sensestrand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety(e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotidemoiety such as inverted thymidine) is present at the 5′-end, the 3′-end,or both of the 5′ and 3′ ends of the sense strand. In anotherembodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a myostatin gene, wherein a majority of thepyrimidine nucleotides present in the double stranded siNA moleculecomprises a sugar modification, each of the two strands of the siNAmolecule can comprise about 21 nucleotides. In one embodiment, about 21nucleotides of each strand of the siNA molecule are base-paired to thecomplementary nucleotides of the other strand of the siNA molecule. Inanother embodiment, about 19 nucleotides of each strand of the siNAmolecule are base-paired to the complementary nucleotides of the otherstrand of the siNA molecule, wherein at least two 3′ terminalnucleotides of each strand of the siNA molecule are not base-paired tothe nucleotides of the other strand of the siNA molecule. In anotherembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine.In one embodiment, each strand of the siNA molecule is base-paired tothe complementary nucleotides of the other strand of the siNA molecule.In one embodiment, about 19 nucleotides of the antisense strand arebase-paired to the nucleotide sequence of the myostatin RNA or a portionthereof. In one embodiment, about 21 nucleotides of the antisense strandare base-paired to the nucleotide sequence of the myostatin RNA or aportion thereof.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a myostatin gene, wherein one of the strands ofthe double stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence ofmyostatin RNA or a portion thereof, the other strand is a sense strandwhich comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double stranded siNA moleculecomprises a sugar modification, and wherein the 5′-end of the antisensestrand optionally includes a phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a myostatin gene, wherein one of the strands ofthe double stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence ofmyostatin RNA or a portion thereof, the other strand is a sense strandwhich comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double stranded siNA moleculecomprises a sugar modification, and wherein the nucleotide sequence or aportion thereof of the antisense strand is complementary to a nucleotidesequence of the untranslated region or a portion thereof of themyostatin RNA.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a myostatin gene, wherein one of the strands ofthe double stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence ofmyostatin RNA or a portion thereof, wherein the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand, wherein a majority of thepyrimidine nucleotides present in the double stranded siNA moleculecomprises a sugar modification, and wherein the nucleotide sequence ofthe antisense strand is complementary to a nucleotide sequence of themyostatin RNA or a portion thereof that is present in the myostatin RNA.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention in a pharmaceutically acceptable carrieror diluent.

In a non-limiting example, the introduction of chemically modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of an siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of an siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding myostatin andthe sense region can comprise sequence complementary to the antisenseregion. The siNA molecule can comprise two distinct strands havingcomplementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against myostatin inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotidescomprising a backbone modified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring or chemicallymodified, each X and Y is independently O, S, N, alkyl, or substitutedalkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl,O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Zare optionally not all O. In another embodiment, a backbone modificationof the invention comprises a phosphonoacetate and/orthiophosphonoacetate internucleotide linkage (see for example Sheehan etal., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, an siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against myostatin inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula II at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisensestrand, or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotides or non-nucleotides ofFormula II at the 5′-end of the sense strand, the antisense strand, orboth strands. In anther non-limiting example, an exemplary siNA moleculeof the invention can comprise about 1 to about 5 or more (e.g., about 1,2, 3, 4, 5, or more) chemically modified nucleotides or non-nucleotidesof Formula II at the 3′-end of the sense strand, the antisense strand,or both strands.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against myostatin inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula III at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisensestrand, or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotide(s) or non-nucleotide(s) ofFormula III at the 5′-end of the sense strand, the antisense strand, orboth strands. In anther non-limiting example, an exemplary siNA moleculeof the invention can comprise about 1 to about 5 or more (e.g., about 1,2, 3, 4, 5, or more) chemically modified nucleotide or non-nucleotide ofFormula III at the 3′-end of the sense strand, the antisense strand, orboth strands.

In another embodiment, an siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against myostatin inside a cell or reconstituted invitro system, wherein the chemical modification comprises a 5′-terminalphosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo;wherein each Z and W is independently O, S, N, alkyl, substituted alkyl,O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, or acetyl; and wherein W,X, Y and Z are not all O.

In one embodiment, the invention features an siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features an siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of an siNA molecule of the invention,for example an siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against myostatin inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or morephosphorothioate internucleotide linkages. For example, in anon-limiting example, the invention features a chemically modified shortinterfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 ormore phosphorothioate internucleotide linkages in one siNA strand. Inyet another embodiment, the invention features a chemically modifiedshort interfering nucleic acid (siNA) individually having about 1, 2, 3,4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in bothsiNA strands. The phosphorothioate internucleotide linkages can bepresent in one or both oligonucleotide strands of the siNA duplex, forexample in the sense strand, the antisense strand, or both strands. ThesiNA molecules of the invention can comprise one or morephosphorothioate internucleotide linkages at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the sense strand, the antisense strand,or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) consecutive phosphorothioate internucleotide linkages atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine phosphorothioate internucleotide linkages inthe sense strand, the antisense strand, or both strands. In yet anothernon-limiting example, an exemplary siNA molecule of the invention cancomprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) purine phosphorothioate internucleotide linkages in the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features an siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features an siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore (specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features an siNA moleculecomprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotidelinkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of one or both siNA sequence strands. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin one or both siNA sequence strands, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of apyrimidine nucleotide in one or both strands of the siNA molecule cancomprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more including every internucleotide linkage of a purinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage.

In another embodiment, a chemically modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically modified, wherein each strand is about 18 to about 27(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides inlength, wherein the duplex has about 18 to about 23 (e.g., about 18, 19,20, 21, 22, or 23) base pairs, and wherein the chemical modificationcomprises a structure having any of Formulae I-VII. For example, anexemplary chemically modified siNA molecule of the invention comprises aduplex having two strands, one or both of which can be chemicallymodified with a chemical modification having any of Formulae I-VII orany combination thereof, wherein each strand consists of about 21nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang,and wherein the duplex has about 19 base pairs. In another embodiment,an siNA molecule of the invention comprises a single stranded hairpinstructure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40,45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 toabout 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and whereinthe siNA can include a chemical modification comprising a structurehaving any of Formulae I-VII or any combination thereof. For example, anexemplary chemically modified siNA molecule of the invention comprises alinear oligonucleotide having about 42 to about 50 (e.g., about 42, 43,44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically modifiedwith a chemical modification having any of Formulae I-VII or anycombination thereof, wherein the linear oligonucleotide forms a hairpinstructure having about 19 base pairs and a 2-nucleotide 3′-terminalnucleotide overhang. In another embodiment, a linear hairpin siNAmolecule of the invention contains a stem loop motif, wherein the loopportion of the siNA molecule is biodegradable. For example, a linearhairpin siNA molecule of the invention is designed such that degradationof the loop portion of the siNA molecule in vivo can generate a doublestranded siNA molecule with 3′-terminal overhangs, such as 3′-terminalnucleotide overhangs comprising about 2 nucleotides.

In another embodiment, an siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 23(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, or 23) base pairs and a 5′-terminal phosphate group thatcan be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20) base pairs, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically modified siNA molecule of the invention comprises alinear oligonucleotide having about 25 to about 35 (e.g., about 25, 26,27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemicallymodified with one or more chemical modifications having any of FormulaeI-VII or any combination thereof, wherein the linear oligonucleotideforms an asymmetric hairpin structure having about 3 to about 18 (e.g.,about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) basepairs and a 5′-terminal phosphate group that can be chemically modifiedas described herein (for example a 5′-terminal phosphate group havingFormula IV). In one embodiment, an asymmetric hairpin siNA molecule ofthe invention contains a stem loop motif, wherein the loop portion ofthe siNA molecule is biodegradable. In another embodiment, an asymmetrichairpin siNA molecule of the invention comprises a loop portioncomprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 16 to about 25 (e.g., about 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides in length, wherein the sense region is about3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, or 18) nucleotides in length, wherein the sense region and theantisense region have at least 3 complementary nucleotides, and whereinthe siNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically modified siNA molecule of the inventioncomprises an asymmetric double stranded structure having separatepolynucleotide strands comprising sense and antisense regions, whereinthe antisense region is about 18 to about 22 (e.g., about 18, 19, 20,21, or 22) nucleotides in length and wherein the sense region is about 3to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, an siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) basepairs, and wherein the siNA can include a chemical modification, whichcomprises a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically modified siNA molecule ofthe invention comprises a circular oligonucleotide having about 42 toabout 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotidesthat is chemically modified with a chemical modification having any ofFormulae I-VII or any combination thereof, wherein the circularoligonucleotide forms a dumbbell shaped structure having about 19 basepairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R5, R3,R8 or R13 serves as a point of attachment to the siNA molecule of theinvention.

In another embodiment, an siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises Oand is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double stranded siNA moleculeof the invention or to a single stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a chemically modified nucleoside ornon-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of theinvention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends ofan siNA molecule of the invention. For example, chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) can be present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the antisense strand, the sense strand, or both antisense andsense strands of the siNA molecule. In one embodiment, the chemicallymodified nucleoside or non-nucleoside (e.g., a moiety having Formula V,VI or VII) is present at the 5′-end and 3′-end of the sense strand andthe 3′-end of the antisense strand of a double stranded siNA molecule ofthe invention. In one embodiment, the chemically modified nucleoside ornon-nucleoside (e.g., a moiety having Formula V, VI or VII) is presentat the terminal position of the 5′-end and 3′-end of the sense strandand the 3′-end of the antisense strand of a double stranded siNAmolecule of the invention. In one embodiment, the chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) is present at the two terminal positions of the 5′-end and 3′-endof the sense strand and the 3′-end of the antisense strand of a doublestranded siNA molecule of the invention. In one embodiment, thechemically modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the penultimate position of the5′-end and 3′-end of the sense strand and the 3′-end of the antisensestrand of a double stranded siNA molecule of the invention. In addition,a moiety having Formula VII can be present at the 3′-end or the 5′-endof a hairpin siNA molecule as described herein.

In another embodiment, an siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a 3′-3′, 3′-2′,2′-3′, or 5′-5′ configuration, such as the 3′-end, the 5′-end, or bothof the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, an siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, an siNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides),wherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in thesense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) against myostatin inside a cell orreconstituted in vitro system comprising a sense region, wherein one ormore pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides), and an antisenseregion, wherein one or more pyrimidine nucleotides present in theantisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). The sense region and/or the antisenseregion can have a terminal cap modification, such as any modificationdescribed herein or shown in FIG. 10, that is optionally present at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/orantisense sequence. The sense and/or antisense region can optionallyfurther comprise a 3′-terminal nucleotide overhang having about 1 toabout 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhangnucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 ormore) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages. Non-limiting examples of these chemicallymodified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein.In any of these described embodiments, the purine nucleotides present inthe sense region are alternatively 2′-O-methyl purine nucleotides (e.g.,wherein all purine nucleotides are 2′-O-methyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides) and one or more purine nucleotides present in the antisenseregion are 2′-O-methyl purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Also, in any of these embodiments, one or more purine nucleotidespresent in the sense region are alternatively purine ribonucleotides(e.g., wherein all purine nucleotides are purine ribonucleotides oralternately a plurality of purine nucleotides are purineribonucleotides) and any purine nucleotides present in the antisenseregion are 2′-O-methyl purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Additionally, in any of these embodiments, one or more purinenucleotides present in the sense region and/or present in the antisenseregion are alternatively selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g.,wherein all purine nucleotides are selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides or alternately a plurality of purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a Northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against myostatin inside a cell or reconstituted invitro system, wherein the chemical modification comprises a conjugatecovalently attached to the chemically modified siNA molecule.Non-limiting examples of conjugates contemplated by the inventioninclude conjugates and ligands described in Vargeese et al., U.S. Ser.No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein inits entirety, including the drawings. In another embodiment, theconjugate is covalently attached to the chemically modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically modified siNA molecule, or any combination thereof. Inone embodiment, a conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically modifiedsiNA molecule is a polyethylene glycol, human serum albumin, or a ligandfor a cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to chemically modified siNA molecules are described inVargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002incorporated by reference herein. The type of conjugates used and theextent of conjugation of siNA molecules of the invention can beevaluated for improved pharmacokinetic profiles, bioavailability, and/orstability of siNA constructs while at the same time maintaining theability of the siNA to mediate RNAi activity. As such, one skilled inthe art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≧2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has a sequence that comprises a sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid. The target molecule can be any molecule of interest. For example,the aptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628. )

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, an siNA molecule canbe assembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, an siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, an siNA molecule of the invention is a singlestranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single strandedpolynucleotide having complementarity to a target nucleic acid sequence.In another embodiment, the single stranded siNA molecule of theinvention comprises a 5′-terminal phosphate group. In anotherembodiment, the single stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a2′,3′-cyclic phosphate). In another embodiment, the single stranded siNAmolecule of the invention comprises about 19 to about 29 (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. In yetanother embodiment, the single stranded siNA molecule of the inventioncomprises one or more chemically modified nucleotides or non-nucleotidesdescribed herein. For example, all the positions within the siNAmolecule can include chemically modified nucleotides such as nucleotideshaving any of Formulae I-VII, or any combination thereof to the extentthat the ability of the siNA molecule to support RNAi activity in a cellis maintained.

In one embodiment, an siNA molecule of the invention is a singlestranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single strandedpolynucleotide having complementarity to a target nucleic acid sequence,wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), anda terminal cap modification, such as any modification described hereinor shown in FIG. 10, that is optionally present at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the antisense sequence. ThesiNA optionally further comprises about 1 to about 4 or more (e.g.,about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the 3′-end ofthe siNA molecule, wherein the terminal nucleotides can further compriseone or more (e.g., 1, 2, 3, 4 or more) phosphorothioate,phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages,and wherein the siNA optionally further comprises a terminal phosphategroup, such as a 5′-terminal phosphate group. In any of theseembodiments, any purine nucleotides present in the antisense region arealternatively 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides). Also, in any ofthese embodiments, any purine nucleotides present in the siNA (i.e.,purine nucleotides present in the sense and/or antisense region) canalternatively be locked nucleic acid (LNA) nucleotides (e.g., whereinall purine nucleotides are LNA nucleotides or alternately a plurality ofpurine nucleotides are LNA nucleotides). Also, in any of theseembodiments, any purine nucleotides present in the siNA arealternatively 2′-methoxyethyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-methoxyethyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-methoxyethyl purinenucleotides). In another embodiment, any modified nucleotides present inthe single stranded siNA molecules of the invention comprise modifiednucleotides having properties or characteristics similar to naturallyoccurring ribonucleotides. For example, the invention features siNAmolecules including modified nucleotides having a Northern conformation(e.g., Northern pseudorotation cycle, see for example Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Assuch, chemically modified nucleotides present in the single strandedsiNA molecules of the invention are preferably resistant to nucleasedegradation while at the same time maintaining the capacity to mediateRNAi.

In one embodiment, the invention features a method for modulating theexpression of a myostatin gene within a cell comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the myostatin gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate theexpression of the myostatin gene in the cell.

In one embodiment, the invention features a method for modulating theexpression of a myostatin gene within a cell comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the myostatin gene and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequence of the target RNA; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate theexpression of the myostatin gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one myostatin gene within a cell comprising:(a) synthesizing siNA molecules of the invention, which can bechemically modified, wherein one of the siNA strands comprises asequence complementary to RNA of the myostatin genes; and (b)introducing the siNA molecules into a cell under conditions suitable tomodulate the expression of the myostatin genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more myostatin genes within a cell comprising:(a) synthesizing one or more siNA molecules of the invention, which canbe chemically modified, wherein the siNA strands comprise sequencescomplementary to RNA of the myostatin genes and wherein the sense strandsequences of the siNAs comprise sequences identical or substantiallysimilar to the sequences of the target RNAs; and (b) introducing thesiNA molecules into a cell under conditions suitable to modulate theexpression of the myostatin genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one myostatin gene within a cell comprising:(a) synthesizing an siNA molecule of the invention, which can bechemically modified, wherein one of the siNA strands comprises asequence complementary to RNA of the myostatin gene and wherein thesense strand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequences of the target RNAs; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate the expression of the myostatin genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients. In one embodiment, theinvention features a method of modulating the expression of a myostatingene in a tissue explant comprising: (a) synthesizing an siNA moleculeof the invention, which can be chemically modified, wherein one of thesiNA strands comprises a sequence complementary to RNA of the myostatingene; and (b) introducing the siNA molecule into a cell of the tissueexplant derived from a particular organism under conditions suitable tomodulate the expression of the myostatin gene in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate the expression ofthe myostatin gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a myostatin gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the myostatin gene and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequence of the target RNA; and (b) introducing the siNAmolecule into a cell of the tissue explant derived from a particularorganism under conditions suitable to modulate the expression of themyostatin gene in the tissue explant. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate the expression of the myostatin gene in thatorganism.

In another embodiment, the invention features a method of modulating theexpression of more than one myostatin gene in a tissue explantcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically modified, wherein one of the siNA strands comprises asequence complementary to RNA of the myostatin genes; and (b)introducing the siNA molecules into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the myostatin genes in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of themyostatin genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a myostatin gene in a subject or organism comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the myostatin gene; and (b) introducing the siNAmolecule into the subject or organism under conditions suitable tomodulate the expression of the myostatin gene in the subject ororganism. The level of myostatin protein or RNA can be determined usingvarious methods well-known in the art.

In another embodiment, the invention features a method of modulating theexpression of more than one myostatin gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically modified, wherein one of the siNA strands comprises asequence complementary to RNA of the myostatin genes; and (b)introducing the siNA molecules into the subject or organism underconditions suitable to modulate the expression of the myostatin genes inthe subject or organism. The level of myostatin protein or RNA can bedetermined as is known in the art.

In one embodiment, the invention features a method for modulating theexpression of a myostatin gene within a cell comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the myostatin gene; and (b) introducing thesiNA molecule into a cell under conditions suitable to modulate theexpression of the myostatin gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one myostatin gene within a cell comprising:(a) synthesizing siNA molecules of the invention, which can bechemically modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the myostatin gene; and (b)contacting the cell in vitro or in vivo with the siNA molecule underconditions suitable to modulate the expression of the myostatin genes inthe cell.

In one embodiment, the invention features a method of modulating theexpression of a myostatin gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the myostatin gene; and (b) contacting a cellof the tissue explant derived from a particular organism with the siNAmolecule under conditions suitable to modulate the expression of themyostatin gene in the tissue explant. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate the expression of the myostatin gene in thatorganism.

In another embodiment, the invention features a method of modulating theexpression of more than one myostatin gene in a tissue explantcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the myostatin gene; and (b)introducing the siNA molecules into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the myostatin genes in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of themyostatin genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a myostatin gene in a subject or organism comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the myostatin gene; and (b) introducing thesiNA molecule into the subject or organism under conditions suitable tomodulate the expression of the myostatin gene in the subject ororganism.

In another embodiment, the invention features a method of modulating theexpression of more than one myostatin gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the myostatin gene; and (b)introducing the siNA molecules into the subject or organism underconditions suitable to modulate the expression of the myostatin genes inthe subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a myostatin gene in a subject or organism comprisingcontacting the subject or organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of themyostatin gene in the subject or organism.

In one embodiment, the invention features a method for preventing ortreating diseases and conditions associated with muscle atrophy,weakness and/or degeneration, such as muscular dystrophy, myotonicdystrophy, myotonia congentia, poliomyelitis, amyotrophic lateralsclerosis (ALS or Lou Gehrig's disease), Guillain-Barre syndrome, musclewasting (e.g., age or HIV related), sarcopenia, myalgias, myopathies,hypotonis, hypotonia, cachexia, spinal cord injury, or muscle injury ina subject or organism comprising contacting the subject or organism withan siNA molecule of the invention under conditions suitable to modulatethe expression of a myostatin gene in the subject or organism. In oneembodiment, the siNA is administered to the subject or organismprophylactically to prevent, inhibit or reduce muscle atrophy, weaknessand/or degeneration associated with muscular dystrophy, myotonicdystrophy, myotonia congentia, poliomyelitis, amyotrophic lateralsclerosis (ALS or Lou Gehrig's disease), Guillain-Barre syndrome, musclewasting, sarcopenia, myalgias, myopathies, hypotonis, cachexia, spinalcord injury, or muscle injury in the subject or organism.

In one embodiment, the invention features a method for preventing ortreating obesity, diabetes (e.g., type I and type II), and insulinresistance in a subject or organism comprising contacting the subject ororganism with an siNA molecule of the invention under conditionssuitable to modulate the expression of a myostatin gene in the subjector organism.

In one embodiment, the invention features a method for preventing ortreating muscular dystrophy (e.g., Becker's muscular dystrophy, Duchennemuscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdlemuscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonicdystrophy, or myotonia congenita) in a subject or organism comprisingcontacting the subject or organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of amyostatin gene in the subject or organism. In one embodiment, the siNAis administered to the subject or organism prophylactically to prevent,inhibit or reduce muscular atrophy, muscle wasting (e.g., age or HIVrelated), muscle degradation and/or muscle weakness associated withmuscular dystrophy. In one embodiment, the siNA is administered to thesubject or organism therapeutically to prevent, inhibit, reduce, orreverse muscular atrophy, muscle wasting (e.g., age or HIV related),muscle degradation and/or muscle weakness associated with musculardystrophy.

In one embodiment, the invention features a method for preventing ortreating muscle wasting disease or sarcopenia (e.g., age or HIV related)in a subject or organism comprising contacting the subject or organismwith an siNA molecule of the invention under conditions suitable tomodulate the expression of a myostatin gene in the subject or organism.

In one embodiment, the invention features a method for preventing ortreating poliomyelitis in a subject or organism comprising contactingthe subject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of a myostatin gene inthe subject or organism.

In one embodiment, the invention features a method for preventing ortreating amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) ina subject or organism comprising contacting the subject or organism withan siNA molecule of the invention under conditions suitable to modulatethe expression of a myostatin gene in the subject or organism.

In one embodiment, the invention features a method for preventing ortreating Guillain-Barre syndrome in a subject or organism comprisingcontacting the subject or organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of amyostatin gene in the subject or organism.

In one embodiment, the invention features a method for preventing ortreating myalgia (e.g., polymyalgia, fibromyalgia) in a subject ororganism comprising contacting the subject or organism with an siNAmolecule of the invention under conditions suitable to modulate theexpression of a myostatin gene in the subject or organism.

In one embodiment, the invention features a method for preventing ortreating myopathy (e.g., Mitochondrial myopathies, Myotubular Myopathy,Nemaline Myopathy, Multicore Myopathy, Cardiomyopathy, Dermatomyositis,Inclusion Body Myositis) in a subject or organism comprising contactingthe subject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of a myostatin gene inthe subject or organism.

In one embodiment, the invention features a method for preventing ortreating hypotonis or hypotonia in a subject or organism comprisingcontacting the subject or organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of amyostatin gene in the subject or organism.

In one embodiment, the invention features a method for preventing ortreating cachexia in a subject or organism comprising contacting thesubject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of a myostatin gene inthe subject or organism.

In one embodiment, the invention features a method for preventing ortreating muscle atrophy associated with spinal cord injury in a subjector organism comprising contacting the subject or organism with an siNAmolecule of the invention under conditions suitable to modulate theexpression of a myostatin gene in the subject or organism.

In one embodiment, the invention features a method for preventing ortreating muscle injury in a subject or organism comprising contactingthe subject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of a myostatin gene inthe subject or organism.

In one embodiment, the invention features a method for inducing orpromoting muscle hypertrophy in a subject or organism comprisingcontacting the subject or organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of amyostatin gene in the subject or organism.

In one embodiment, the invention features a method for inducing orpromoting muscle strength in a subject or organism comprising contactingthe subject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of a myostatin gene inthe subject or organism.

In one embodiment, the invention features a method for improvingathletic performance in a subject or organism comprising contacting thesubject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of a myostatin gene inthe subject or organism.

In one embodiment, the invention features a method for preventing muscleatrophy in an astronaut subject comprising contacting the subject withan siNA molecule of the invention under conditions suitable to modulatethe expression of a myostatin gene in the subject or organism.

In one embodiment, the invention features a method for inducing musclehyperthropy in livestock organisms (e.g., cattle, swine, and/or poultry)comprising contacting the organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of amyostatin gene in the subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one myostatin genes in a subject or organismcomprising contacting the subject or organism with one or more siNAmolecules of the invention under conditions suitable to modulate theexpression of the myostatin genes in the subject or organism.

The siNA molecules of the invention can be designed to down regulate orinhibit target (e.g., myostatin) gene expression through RNAi targetingof a variety of RNA molecules. In one embodiment, the siNA molecules ofthe invention are used to target various RNAs corresponding to a targetgene. Non-limiting examples of such RNAs include messenger RNA (mRNA),alternate RNA splice variants of target gene(s), post-transcriptionallymodified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNAtemplates. If alternate splicing produces a family of transcripts thatare distinguished by usage of appropriate exons, the instant inventioncan be used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, pharmaceutical discoveryapplications, molecular diagnostic and gene function applications, andgene mapping, for example using single nucleotide polymorphism mappingwith siNA molecules of the invention. Such applications can beimplemented using known gene sequences or from partial sequencesavailable from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as myostatin family genes. As such, siNA moleculestargeting multiple myostatin targets can provide increased therapeuticeffect. In addition, siNA can be used to characterize pathways of genefunction in a variety of applications. For example, the presentinvention can be used to inhibit the activity of target gene(s) in apathway to determine the function of uncharacterized gene(s) in genefunction analysis, mRNA function analysis, or translational analysis.The invention can be used to determine potential target gene pathwaysinvolved in various diseases and conditions toward pharmaceuticaldevelopment. The invention can be used to understand pathways of geneexpression involved in, for example, muscle hypertrophy, muscle atrophy,degradation, or weakness.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession Nos., for example, myostatin genes encoding RNAsequence(s) referred to herein by Genbank Accession number, for example,Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 19 to about 25 (e.g., about 19, 20, 21,22, 23, 24, or 25) nucleotides in length. In one embodiment, the assaycan comprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. In another embodiment, fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, Northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4^(N), where N represents thenumber of base paired nucleotides in each of the siNA construct strands(e.g., for an siNA construct having 21 nucleotide sense and antisensestrands with 19 base pairs, the complexity would be 4¹⁹); and (b)assaying the siNA constructs of (a) above, under conditions suitable todetermine RNAi target sites within the target myostatin RNA sequence. Inanother embodiment, the siNA molecules of (a) have strands of a fixedlength, for example about 23 nucleotides in length. In yet anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 19 to about 25 (e.g., about 19, 20, 21,22, 23, 24, or 25) nucleotides in length. In one embodiment, the assaycan comprise a reconstituted in vitro siNA assay as described in Example7 herein. In another embodiment, the assay can comprise a cell culturesystem in which target RNA is expressed. In another embodiment,fragments of myostatin RNA are analyzed for detectable levels ofcleavage, for example, by gel electrophoresis, Northern blot analysis,or RNAse protection assays, to determine the most suitable targetsite(s) within the target myostatin RNA sequence. The target myostatinRNA sequence can be obtained as is known in the art, for example, bycloning and/or transcription for in vitro systems, and by cellularexpression in in vivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 19 to about25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. Inone embodiment, the assay can comprise a reconstituted in vitro siNAassay as described herein. In another embodiment, the assay can comprisea cell culture system in which target RNA is expressed. Fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, Northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by an siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention, which can be chemically modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease, trait or condition in a subject, comprising administering tothe subject a composition of the invention under conditions suitable forthe treatment or prevention of the disease, trait, or condition in thesubject, alone or in conjunction with one or more other therapeuticcompounds. In yet another embodiment, the invention features a methodfor preventing or treating myopathic diseases, injuries, or conditionsin a subject comprising administering to the subject a composition ofthe invention under conditions suitable for the prevention or treatmentof the myopathic disease, injury, or condition in the subject.

In another embodiment, the invention features a method for validating amyostatin gene target, comprising: (a) synthesizing an siNA molecule ofthe invention, which can be chemically modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a myostatin targetgene; (b) introducing the siNA molecule into a cell, tissue, or organismunder conditions suitable for modulating expression of the myostatintarget gene in the cell, tissue, or organism; and (c) determining thefunction of the gene by assaying for any phenotypic change in the cell,tissue, or organism.

In another embodiment, the invention features a method for validating amyostatin target comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a myostatin targetgene; (b) introducing the siNA molecule into a biological system underconditions suitable for modulating expression of the myostatin targetgene in the biological system; and (c) determining the function of thegene by assaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell,tissue, or organism, or extract thereof. The term biological system alsoincludes reconstituted RNAi systems that can be used in an in vitrosetting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, apoptosis, proliferation,motility, protein expression or RNA expression or other physical orchemical changes as can be assayed by methods known in the art. Thedetectable change can also include expression of reportergenes/molecules such as Green Florescent Protein (GFP) or various tagsthat are used to identify an expressed protein or any other cellularcomponent that can be assayed.

In one embodiment, the invention features a kit containing an siNAmolecule of the invention, which can be chemically modified, that can beused to modulate the expression of a myostatin target gene in abiological system, including, for example, in a cell, tissue, ororganism. In another embodiment, the invention features a kit containingmore than one siNA molecule of the invention, which can be chemicallymodified, that can be used to modulate the expression of more than onemyostatin target gene in a biological system, including, for example, ina cell, tissue, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically modified. Inanother embodiment, the cell containing an siNA molecule of theinvention is a mammalian cell. In yet another embodiment, the cellcontaining an siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of an siNA molecule of the invention,which can be chemically modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing ansiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for example,under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double stranded siNA molecule.

In another embodiment, the invention features a method for synthesizingan siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example, under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating the doublestranded siNA molecule, for example using a trityl-on synthesis strategyas described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against myostatin, wherein the siNA construct comprises one or morechemical modifications, for example, one or more chemical modificationshaving any of Formulae I-VII or any combination thereof that increasesthe nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In one embodiment, the invention features siNA constructs that mediateRNAi against myostatin, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the sense and antisense strands of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoan siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against myostatin, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against myostatin, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against myostatin, wherein the siNA construct comprises one or morechemical modifications described herein that modulate the polymeraseactivity of a cellular polymerase capable of generating additionalendogenous siNA molecules having sequence homology to the chemicallymodified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically modified siNAmolecule.

In one embodiment, the invention features chemically modified siNAconstructs that mediate RNAi against myostatin in a cell, wherein thechemical modifications do not significantly effect the interaction ofsiNA with a target RNA molecule, DNA molecule and/or proteins or otherfactors that are essential for RNAi in a manner that would decrease theefficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against myostatin comprising(a) introducing nucleotides having any of Formula I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against myostatintarget RNA comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against myostatintarget DNA comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against myostatin, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the cellularuptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against myostatin with improved cellular uptakecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against myostatin, wherein the siNA construct comprises one or morechemical modifications described herein that increases thebioavailability of the siNA construct, for example, by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siNA construct, or by attachingconjugates that target specific tissue types or cell types in vivo.Non-limiting examples of such conjugates are described in Vargeese etal., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability comprising (a)introducing a conjugate into the structure of an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. Such design or modifications are expected to enhance theactivity of siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of an siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, an siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”,“Stab 9/22”, “Stab 23/24”, and “Stab 24/25” chemistries and variantsthereof (see Table IV) wherein the 5′-end and 3′-end of the sense strandof the siNA do not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of an siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 17/22”,“Stab 23/24”, and “Stab 24/25” chemistries and variants thereof (seeTable IV) wherein the 5′-end and 3′-end of the sense strand of the siNAdo not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g. chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b). In one embodiment, the method further comprisesre-screening the chemically modified siNA molecules of step (c) underconditions suitable for isolating chemically modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screeningchemically modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include an siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 4-6, and Tables IIand III herein. For example the siNA can be a double strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 19 base pairs); the antisense strand comprises nucleotide sequencethat is complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense strand comprises nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. Alternatively, the siNA is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions of the siNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s). The siNA can be a polynucleotide witha duplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The siNA can be a circular single stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siNA molecule capable of mediating RNAi. The siNA can alsocomprise a single stranded polynucleotide having nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide, chemicallymodified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), andothers. In addition, as used herein, the term RNAi is meant to beequivalent to other terms used to describe sequence specific RNAinterference, such as post transcriptional gene silencing, translationalinhibition, or epigenetics. For example, siNA molecules of the inventioncan be used to epigenetically silence genes at both thepost-transcriptional level and the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation pattern to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237).

In one embodiment, an siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCTApplication No. US04/16390, filed May 24, 2004).

In one embodiment, an siNA molecule of the invention is amultifunctional siNA, (see for example FIGS. 16-22 and Jadhav et al.,U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and International PCTApplication No. US04/16390, filed May 24, 2004). The multifunctionalsiNA of the invention can comprise sequence targeting, for example, tworegions of myostatin RNA (see for example target sequences in Tables IIand III).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 19 to about 22, or about 19, 20, 21, or 22 nucleotides) anda loop region comprising about 4 to about 8 (e.g., about 4, 5, 6, 7, or8) nucleotides, and a sense region having about 3 to about 18 (e.g.,about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18)nucleotides that are complementary to the antisense region. Theasymmetric hairpin siNA molecule can also comprise a 5′-terminalphosphate group that can be chemically modified. The loop portion of theasymmetric hairpin siNA molecule can comprise nucleotides,non-nucleotides, linker molecules, or conjugate molecules as describedherein.

By “asymmetric duplex” as used herein is meant an siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system e.g. about 19 to about 22 (e.g. about 19, 20,21, or 22) nucleotides and a sense region having about 3 to about 18(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18)nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. In oneembodiment, inhibition, down regulation, or reduction of gene expressionis associated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g. RNA) or inhibition oftranslation. In one embodiment, inhibition, down regulation, orreduction of gene expression is associated with pretranscriptionalsilencing.

By “gene”, or “target gene”, is meant, a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for siNAmediated RNA interference in modulating the activity of fRNA or ncRNAinvolved in functional or regulatory cellular processes. Aberrant fRNAor ncRNA activity leading to disease can therefore be modulated by siNAmolecules of the invention. siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of anorganism or cell, by intervening in cellular processes such as geneticimprinting, transcription, translation, or nucleic acid processing(e.g., transamination, methylation etc.). The target gene can be a genederived from a cell, an endogenous gene, a transgene, or exogenous genessuch as genes of a pathogen, for example a virus, which is present inthe cell after infection thereof. The cell containing the target genecan be derived from or contained in any organism, for example a plant,animal, protozoan, virus, bacterium, or fungus. Non-limiting examples ofplants include monocots, dicots, or gymnosperms. Non-limiting examplesof animals include vertebrates or invertebrates. Non-limiting examplesof fungi include molds or yeasts. For a review, see for example Snyderand Gerstein, 2003, Science, 300, 258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl,GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-aminosymmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3,AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AUamino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CCcarbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GAN3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GGamino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GUcarbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl,UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3,GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “myostatin” as used herein is meant, any growth/differentiationfactor 8 (GDF8) protein, peptide, or polypeptide having GDF8/myostatinactivity (e.g., control and maintenance of muscle mass), such as encodedby Genbank Accession Nos. shown in Table I. The term myostatin alsorefers to nucleic acid sequences encoding any GDF8/myostatin protein,peptide, or polypeptide having GDF8 myostatin activity, such as controland maintenance of muscle mass. The term “myostatin” is also meant toinclude other myostatin sequences, such as other growth differentiationfactor isoforms, mutant GDF8/myostatin genes, splice variants ofGDF8/myostatin genes, and/or GDF8/myostatin gene polymorphisms.

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system or organism to anotherbiological system or organism. The polynucleotide can include bothcoding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of an siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of an siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence.

In one embodiment, siNA molecules of the invention that down regulate orreduce myostatin gene expression are used for treating diseases andconditions associated with muscular atrophy, muscle weakness, muscledysfunction, or muscle destruction, including muscular dystrophy,myotonic dystrophy, myotonia congentia, poliomyelitis, amyotrophiclateral sclerosis (ALS or Lou Gehrig's disease), Guillain-Barresyndrome, muscle wasting, sarcopenia, myalgias, myopathies, hypotonis,hypotonia, cachexia, spinal cord injury, or muscle injury, oralternately for muscle hypertrophy, including use for increasedstrength, athleticism, bodybuilding, prevention of muscle atrophy (e.g.,in astronauts), or cosmetic applications in a subject or organism.

In one embodiment, siNA molecules of the invention that down regulate orreduce myostatin gene expression are used for treating or preventingobesity, diabetes (e.g., type I or type II), cardiovascular disease,and/or insulin resistance in a subject or organism.

By “muscular dystrophy” is meant any disease, disorder, or conditioncharacterized by dystrophic loss of muscle mass or function, includingBecker's muscular dystrophy, Duchenne muscular dystrophy,facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy,Emery-Dreifuss muscular dystrophy, myotonic dystrophy, and/or myotoniacongenita.

By “spinal cord injury” is meant, any injury to the spinal cord,including traumatic, degenerative or infectious spinal cord injuriesinvolving inflammation, compression, tearing, severing, shearing,mechanical disruption, transection, extradural pathology, or distractionof neural elements of the spinal cord and resulting motor deficitsresulting from such injury. The term “spinal cord injury” or “SCI” alsoencompasses anterior cord syndrome, Brown-Séquard syndrome, central cordsyndrome, conus medullaris syndrome, and cauda equina syndrome andinfectious conditions such as meningitis, infections involving thespinal canal including epidural abscesses (infection in the epiduralspace), meningitis (infection of the meninges), subdural abscesses(infections of the subdural space), and intramedullary abscesses(infections within the spinal cord).

In one embodiment of the present invention, each sequence of an siNAmolecule of the invention is independently about 18 to about 24nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22,23, or 24 nucleotides in length. In another embodiment, the siNAduplexes of the invention independently comprise about 17 to about 23base pairs (e.g., about 17, 18, 19, 20, 21, 22, or 23). In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 16to about 22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs.Exemplary siNA molecules of the invention are shown in Table II.Exemplary synthetic siNA molecules of the invention are shown in TableIII and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues (e.g., scalp hair follicles). Thenucleic acid or nucleic acid complexes can be locally administered torelevant tissues ex vivo, or in vivo through direct dermal application,transdermal application, or injection, with or without theirincorporation in biopolymers. In particular embodiments, the nucleicacid molecules of the invention comprise sequences shown in TablesII-III and/or FIGS. 4-5. Examples of such nucleic acid molecules consistessentially of sequences defined in these tables and figures.Furthermore, the chemically modified constructs described in Table IVcan be applied to any siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribofuranose moiety. The terms includedouble stranded RNA, single stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used forpreventing or treating diseases and conditions associated with muscularatrophy, muscle weakness, muscle dysfunction, or muscle destruction,including muscular dystrophy, myotonic dystrophy, myotonia congentia,poliomyelitis, amyotrophic lateral sclerosis (ALS or Lou Gehrig'sdisease), Guillain-Barre syndrome, muscle wasting (e.g., age or HIVrelated), sarcopenia, myalgias, myopathies, hypotonis, hypotonia,cachexia, spinal cord injury, or muscle injury in a subject or organism.The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used forpreventing or treating obesity, diabetes, cardiovascular disease, orinsulin resistance in a subject or organism. Alternately the nucleicacid molecules of the instant invention, individually, or in combinationor in conjunction with other drugs, can be used for promoting musclehypertrophy, including use for increased strength, athleticism,bodybuilding, prevention of muscle atrophy (e.g., in astronauts), orcosmetic applications in a subject or organism. For example, the siNAmolecules can be administered to a subject or can be administered toother appropriate cells evident to those skilled in the art,individually or in combination with one or more drugs under conditionssuitable for the treatment.

In one embodiment, the siNA molecules can be used in combination withother known treatments to prevent or treat diseases and conditionsassociated with muscular atrophy, muscle weakness, muscle dysfunction,or muscle destruction, including muscular dystrophy, myotonic dystrophy,myotonia congentia, poliomyelitis, amyotrophic lateral sclerosis (ALS orLou Gehrig's disease), Guillain-Barre syndrome, muscle wasting,sarcopenia, myalgias, myopathies, hypotonis, hypotonia, cachexia, spinalcord injury, or muscle injury in a subject or organism. In oneembodiment, the siNA molecules can be used in combination with otherknown treatments to treat obesity, diabetes, cardiovascular disease, orinsulin resistance in a subject or organism. Alternately the siNAmolecules can be used in combination with other known treatments topromote muscle hypertrophy, including use for increased strength,athleticism, bodybuilding, prevention of muscle atrophy (e.g., inastronauts), or cosmetic applications in a subject or organism. Forexample, the described molecules could be used in combination with oneor more known compounds, treatments, or procedures to promote ormaintain muscle hypertrophy or muscle growth as are known in the art,including anabolic and androgenic steroid compounds and/or growthfactors.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of an siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms an siNA molecule. Non-limitingexamples of such expression vectors are described in Paul et al., 2002,Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, NatureBiotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500;and Novina et al., 2002, Nature Medicine, advance online publicationdoi:10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for an siNA molecule having complementarity to aRNA molecule referred to by a Genbank Accession numbers, for exampleGenbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form ansiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs. Theantisense strand of constructs A-F comprise sequence complementary toany target nucleic acid sequence of the invention. Furthermore, when aglyceryl moiety (L) is present at the 3′-end of the antisense strand forany construct shown in FIG. 4A-F, the modified internucleotide linkageis optional.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s”, optionally connects the (N N)nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target RNAsequence, and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 5A-F shows non-limiting examples of specific chemically modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to a myostatin siNA sequence. Such chemicalmodifications can be applied to any myostatin sequence and/or myostatinpolymorphism sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined myostatin target sequence, wherein the senseregion comprises, for example, about 19, 20, 21, or 22 nucleotides (N)in length, which is followed by a loop sequence of defined sequence (X),comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in an siNA transcript having specificity for a myostatintarget sequence and having self-complementary sense and antisenseregions.

FIG. 7C: The construct is heated (for example to about 95° C.) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example, by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined myostatin target sequence, wherein the senseregion comprises, for example, about 19, 20, 21, or 22 nucleotides (N)in length, and which is followed by a 3′-restriction site (R2) which isadjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palindrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palindrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allow the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of target gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 16A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 16B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 17A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 17B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifunctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 18Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 18B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifunctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 19A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 19B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example, a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC complex to initiate RNAinterference mediated cleavage of its corresponding target. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target region. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

DETAILED DESCRIPTION OF THE INVENTION

Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or an siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing an siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of an siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

An siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to an siNA molecule of the invention or thesense and antisense strands of an siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example, enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules). The treatment ofsubjects with siNA molecules can also include combinations of differenttypes of nucleic acid molecules, such as enzymatic nucleic acidmolecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate,decoys, and aptamers.

In another aspect an siNA molecule of the invention comprises one ormore 5′ and/or a 3′-cap structure, for example, on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of cap moieties areshown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and suitable heterocyclic groups include furanyl, thienyl, pyridyl,pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl andthe like, all optionally substituted. An “amide” refers to an—C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An“ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylarylor hydrogen.

“Nucleotide” as used herein, and as recognized in the art, includesnatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

An siNA molecule of the invention can be adapted for use to treat orprevent diseases, traits, or conditions associated with muscularatrophy, muscle weakness, muscle dysfunction, or muscle destruction,including muscular dystrophy, myotonic dystrophy, myotonia congentia,poliomyelitis, amyotrophic lateral sclerosis (ALS or Lou Gehrig'sdisease), Guillain-Barre syndrome, muscle wasting (e.g., age or HIVrelated), sarcopenia, myalgias, myopathies, hypotonis, hypotonia,cachexia, spinal cord injury, or muscle injury, or any other relatedtrait, disease or condition that is related to or will respond to thelevels of myostatin in a cell or tissue, alone or in combination withother therapies. Nucleic acid molecules of the invention can also beadapted to treat or prevent obesity, diabetes (e.g., type I and typeII), and insulin resistance in a subject. Alternately the nucleic acidmolecules of the instant invention, individually, or in combination orin conjunction with other drugs, can be adapted for use to promotemuscle hypertrophy, including use for increased strength, athleticism,bodybuilding, prevention of muscle atrophy (e.g., in astronauts), orcosmetic applications in a subject or organism. For example, an siNAmolecule can comprise a delivery vehicle, including liposomes, foradministration to a subject, carriers and diluents and their salts,and/or can be present in pharmaceutically acceptable formulations.Methods for the delivery of nucleic acid molecules are described inAkhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies forAntisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al.,1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb.Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser.,752, 184-192, all of which are incorporated herein by reference.Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO94/02595 further describe the general methods for delivery of nucleicacid molecules. These protocols can be utilized for the delivery ofvirtually any nucleic acid molecule. Nucleic acid molecules can beadministered to cells by a variety of methods known to those of skill inthe art, including, but not restricted to, encapsulation in liposomes,by iontophoresis, or by incorporation into other vehicles, such asbiodegradable polymers, hydrogels, cyclodextrins (see for exampleGonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al.,International PCT publication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In another embodiment,the nucleic acid molecules of the invention can also be formulated orcomplexed with polyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives.

In one embodiment, an siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, an siNA molecule of the invention is complexed withdelivery systems as described in U.S. Patent Application Publication No.2003077829 and International PCT Publication Nos. WO 00/03683 and WO02/087541, all incorporated by reference herein in their entiretyincluding the drawings.

In one embodiment, the invention features the use of methods to deliverthe nucleic acid molecules of the instant invention to muscle tissue.Non limiting examples of such methods as are known in the art aredescribed for example in Wells et al., 2003, FEBS Lett., 552, 145-9;Murakami et al., 2003, Muscle Nerve., 27, 237-41; Lu et al., 2003,Nature Medicine., 9, 1009-14; Rando et al., 2000, PNAS, 97, 5363-8; andGollins et al., 2003, Gene Ther., 10, 504-12; Yuasa et al., 2002, GeneTher., 23, 1576-88; Liu et al, 2001, Mol Ther., 4, 45-51; and Fassati etal., 1997, J Clin Invest., 100, 620-8.

In one embodiment, dermal delivery systems of the invention include, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and nonaqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer. Examples of liposomes which can be used in thisinvention include the following: (1) CellFectin, 1:1.5 (M/M) liposomeformulation of the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-|1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PharmaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, an siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886;U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No.5,138,045, all incorporated by reference herein.

In one embodiment, the invention features a pharmaceutical compositioncomprising one or more nucleic acid(s) of the invention in an acceptablecarrier, such as a stabilizer, buffer, and the like. The polynucleotidesof the invention can be administered (e.g., RNA, DNA or protein) andintroduced to a subject by any standard means, with or withoutstabilizers, buffers, and the like, to form a pharmaceuticalcomposition. When it is desired to use a liposome delivery mechanism,standard protocols for formation of liposomes can be followed. Thecompositions of the present invention can also be formulated and used ascreams, gels, sprays, oils and other suitable compositions for topical,dermal, or transdermal administration as is known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemic orlocal administration, into a cell or subject, including for example ahuman. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to asubject by systemic administration in a pharmaceutically acceptablecomposition or formulation. By “systemic administration” is meant invivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes that lead to systemic absorption include, without limitation:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexposes the siNA molecules of the invention to an accessible diseasedtissue. The rate of entry of a drug into the circulation has been shownto be a function of molecular weight or size. The use of a liposome orother drug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation that can facilitate the association of drug withthe surface of cells, such as, lymphocytes and macrophages is alsouseful. This approach can provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of the nucleic acid molecules ofthe instant invention in the physical location most suitable for theirdesired activity. Non-limiting examples of agents suitable forformulation with the nucleic acid molecules of the instant inventioninclude: P-glycoprotein inhibitors (such as Pluronic P85); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery (Emerich, D F et al, 1999, Cell Transplant,8, 47-58); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono-or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pats.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intra-muscular administration, by administration totarget cells ex-planted from a subject followed by reintroduction intothe subject, or by any other means that would allow for introductioninto the desired target cell (for a review see Couture et al., 1996,TIG., 12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of ansiNA duplex, or a single self-complementary strand that self hybridizesinto an siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi:10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of an siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of an siNA molecule, wherein the sequenceis operably linked to the 3′-end of the open reading frame and whereinthe sequence is operably linked to the initiation region, the intron,the open reading frame and the termination region in a manner whichallows expression and/or delivery of the siNA molecule.

Myostatin Biology and Biochemistry

Myostatin, also known as MSTN, Growth/Differentiation Factor 8, andGDF8, is a member of the transforming growth factor-beta superfamilywhich encompasses a large number of growth and differentiation factorsthat play important roles in regulating embryonic development and inmaintaining tissue homeostasis in adult animals. Myostatin is a memberof this superfamily with a role in the control and maintenance ofskeletal muscle mass. The myostatin gene is expressed specifically indeveloping and adult skeletal muscle. The gene encodes a 376-amino acidpolypeptide that contains all the sequence hallmarks of the TGF-betasuperfamily. During early stages of embryogenesis, myostatin expressionis restricted to the myotome compartment of developing somites. At laterstages and in adult animals, myostatin is expressed in many differentmuscles throughout the body. Myostatin is transcribed as a 3.1-kb mRNAspecies that encodes a 335-amino acid precursor protein. Myostatin isexpressed uniquely in human skeletal muscle as a 26-kD matureglycoprotein (myostatin-immunoreactive protein) and is secreted into theplasma. Myostatin immunoreactivity is detectable in human skeletalmuscle in both type 1 and type 2 muscle fibers.

Several investigations have studied the role of myostatin in musclemodeling. Myostatin expression correlates inversely with fat-free massin humans. Increased expression of the myostatin gene is associated withweight loss in men with AIDS wasting syndrome. Gonzalez-Cadavid et al.,1998, PNAS, 95, 14938-14943 examined the expression of myostatin inskeletal muscle and serum of healthy and HIV-infected men. The serum andintramuscular concentrations of myostatin-immunoreactive protein wereincreased in HIV-infected men with weight loss compared with healthy menand correlated inversely with fat-free mass index. Zimmers et al., 2002,Science, 296, 1486-1488, induced cachexia in mice by systemicallyadministered myostatin. Systemic overexpression of myostatin in adultmice was found to induce profound muscle and fat loss without diminutionof nutrient intake. This effect is similar to that seen in humancachexia syndromes, and suggests that myostatin can be a usefulpharmacologic target in clinical settings such as cachexia, where musclegrowth is desired. Schuelke et al., 2004, N. Engl. J. Med., 350,2682-88, describe a young male with a loss of function mutation in themyostatin gene characterized by profound muscle hypertrophy andincreased strength compared to similar aged subjects or the generalpopulation.

The role of myostatin has been studied in various animals. McPerron etal., 1997, Nature, 387, 83-90, disrupted the myostatin gene by genetargeting in mice. Myostatin-null animals were significantly larger thanwildtype animals and showed a large and widespread increase in skeletalmuscle mass. Individual muscles of mutant animals weighed 2 to 3 timesmore than those of wildtype animals, and the increase in mass appearedto result from a combination of muscle cell hyperplasia and hypertrophy.The authors suggested that myostatin functions specifically as anegative regulator of skeletal muscle growth. Furthermore, Lin et al.,2002, Biochem. Biophys. Res. Commun., 291, 701-706, observed increasedskeletal muscle mass in a myostatin-null mouse model compared towildtype animals as early as 4 weeks of age. The mutant mice showedreduced production and secretion of leptin which was associated withreduced fat deposition. The reduced adipogenesis in the knockout micesuggests that myostatin is involved in regulating adiposity as well asmuscularity. Several cattle breeds have been observed to showexceptional muscle development commonly referred to as ‘double-muscled.’Double-muscled animals are characterized by an increase in muscle massof about 20%, due to general skeletal muscle hyperplasia, or an increasein the number of muscle fibers rather than in their individual diameter.Grobet et al., 1997, Nature Genet. 17, 71-74, used a positionalcandidate approach to demonstrate that a mutation in the bovine genewhich encodes myostatin is responsible for the double-muscled phenotype.The authors found an 11-bp deletion in the coding sequence for thebioactive C-terminal domain of the protein causing the muscularhypertrophy in these animals.

Bogdanovich et al., 2002, Nature, 420, 418-421, tested the ability ofinhibition of myostatin in vivo to ameliorate the dystrophic phenotypein the mdx mouse model of Duchenne muscular dystrophy (DMD). The authorsblocked endogenous myostatin in mdx mice by intraperitoneal injectionsof blocking antibodies for 3 months and found increase in body weight,muscle mass, muscle size, and absolute muscle strength along with asignificant decrease in muscle degeneration and concentrations of serumcreatine kinase. The authors concluded that myostatin blockade providesa novel, pharmacologic strategy for treatment of diseases associatedwith muscle wasting such as DMD, and circumvents the major problemsassociated with conventional gene therapy in these disorders.Furthermore, in myostatin null mice (Mstn −/−) crossed with mdx mice, amodel for Duchenne and Becker muscular dystrophy, Wagner et al., 2002,Ann. Neurol. 52, 832-836, found increased muscle mass, increased bodyweight, increased muscle fiber size, and increased strength compared toMstn +/+/mdx mice. There was also a reduction in the extent of musclefibrosis in these mice. The authors noted that although the loss ofmyostatin does not correct the primary defect in the mdx mice, it mayameliorate some features of the dystrophic phenotype.

Based upon the current understanding of myostatin, the modulation ofmyostatin and related genes is instrumental in the development of newtreatments for diseases, traits and conditions associated with muscularatrophy, muscle weakness, muscle dysfunction, or muscle destruction,such as muscular dystrophy, myotonic dystrophy, myotonia congentia,poliomyelitis, amyotrophic lateral sclerosis (ALS or Lou Gehrig'sdisease), Guillain-Barre syndrome, muscle wasting, sarcopenia, myalgias,myopathies, hypotonis, hypotonia, cachexia, spinal cord injury, ormuscle injury. Nucleic acid molecules of the invention are also usefulin treating or preventing obesity, diabetes (e.g., type I and type II),and insulin resistance. As such, there exists a need for selectiveinhibitors for myostatin in this regard. Modulation of myostatin usingsmall interfering nucleic acid (siNA) mediated RNAi represents a novelapproach to the treatment and prevention of such diseases, traits, andconditions, and for other applications that benefit from musclehypertrophy, such as for use for increased strength, athleticism,bodybuilding, prevention of muscle atrophy (e.g., in astronauts), orcosmetic applications in a subject or organism, or for generatingimproved livestock.

Examples

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of an siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example, using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H2O followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Tables II and III). Ifterminal TT residues are desired for the sequence (as described inparagraph 7), then the two 3′ terminal nucleotides of both the sense andantisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the target RNA sequence.

10. Other design considerations can be used when selecting targetnucleic acid sequences, see, for example, Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004,doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32,doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to amyostatin target sequence is used to screen for target sites in cellsexpressing myostatin RNA, such as C2C12 cells. The general strategy usedin this approach is shown in FIG. 9. A non-limiting example of such aspool is a pool comprising sequences having SEQ ID NOs. 1-436. CulturedC2C12 cells are transfected with the pool of siNA constructs and cellsthat demonstrate a phenotype associated with myostatin inhibition aresorted. The pool of siNA constructs can be expressed from transcriptioncassettes inserted into appropriate vectors (see for example FIG. 7 andFIG. 8). The siNA from cells demonstrating a positive phenotypic change(e.g., decreased proliferation, decreased myostatin mRNA levels ordecreased myostatin protein expression), are sequenced to determine themost suitable target site(s) within the target myostatin RNA sequence.

Example 4 Myostatin Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the myostatinRNA target and optionally prioritizing the target sites on the basis offolding (structure of any given sequence analyzed to determine siNAaccessibility to the target), by using a library of siNA molecules asdescribed in Example 3, or alternately by using an in vitro siNA systemas described in Example 6 herein. siNA molecules were designed thatcould bind each target and are optionally individually analyzed bycomputer folding to assess whether the siNA molecule can interact withthe target sequence. Varying the length of the siNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplementary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siNA duplexes or varying length or basecomposition. By using such methodologies, siNA molecules can be designedto target sites within any known RNA sequence, for example those RNAsequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a rib nucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Deprotection and purification of the siNA can beperformed as is generally described in Usman et al., U.S. Pat. No.5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellonet al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No.6,303,773, or Scaringe supra, incorporated by reference herein in theirentireties. Additionally, deprotection conditions can be modified toprovide the best possible yield and purity of siNA constructs. Forexample, applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi in Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting myostatin RNA targets. The assaycomprises the system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with myostatin target RNA. A Drosophila extract derivedfrom syncytial blastoderm is used to reconstitute RNAi activity invitro. Target RNA is generated via in vitro transcription from anappropriate myostatin expressing plasmid using T7 RNA polymerase or viachemical synthesis as described herein. Sense and antisense siNA strands(for example 20 uM each) are annealed by incubation in buffer (such as100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesiumacetate) for 1 minute at 90° C. followed by 1 hour at 37° C., thendiluted in lysis buffer (for example 100 mM potassium acetate, 30 mMHEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitoredby gel electrophoresis on an agarose gel in TBE buffer and stained withethidium bromide. The Drosophila lysate is prepared using zero totwo-hour-old embryos from Oregon R flies collected on yeasted molassesagar that are dechorionated and lysed. The lysate is centrifuged and thesupernatant isolated. The assay comprises a reaction mixture containing50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10%[vol/vol] lysis buffer containing siNA (10 nM final concentration). Thereaction mixture also contains 10 mM creatine phosphate, 10 ug/mlcreatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP,5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. Thefinal concentration of potassium acetate is adjusted to 100 mM. Thereactions are pre-assembled on ice and preincubated at 25° C. for 10minutes before adding RNA, then incubated at 25° C. for an additional 60minutes. Reactions are quenched with 4 volumes of 1.25× Passive LysisBuffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis orother methods known in the art and are compared to control reactions inwhich siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed as idescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without siNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites in themyostatin RNA target for siNA mediated RNAi cleavage, wherein aplurality of siNA constructs are screened for RNAi mediated cleavage ofthe myostatin RNA target, for example, by analyzing the assay reactionby electrophoresis of labeled target RNA, or by Northern blotting, aswell as by other methodology well known in the art.

Example 7 Nucleic Acid Inhibition of Myostatin Target RNA in Vitro

siNA molecules targeted to the human myostatin RNA are designed andsynthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure. The target sequences and the nucleotide location within themyostatin RNA are given in Table II and III.

Two formats are used to test the efficacy of siNAs targeting myostatin.First, the reagents are tested in cell culture, for example using C2C12cells to determine the extent of RNA and protein inhibition. siNAreagents (e.g.; see Tables II and III) are selected against themyostatin target as described herein. RNA inhibition is measured afterdelivery of these reagents by a suitable transfection agent to, forexample, C2C12 cells. Relative amounts of target RNA are measured versusactin using real-time PCR monitoring of amplification (e.g., ABI 7700Taqman®). A comparison is made to a mixture of oligonucleotide sequencesmade to unrelated targets or to a randomized siNA control with the sameoverall length and chemistry, but randomly substituted at each position.Primary and secondary lead reagents are chosen for the target andoptimization performed. After an optimal transfection agentconcentration is chosen, a RNA time-course of inhibition is performedwith the lead siNA molecule. In addition, a cell-plating format can beused to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., C2C12) are seeded, for example, at 1×10⁵ cells per well ofa six-well dish in EGM-2 (BioWhittaker) the day before transfection.siNA (final concentration, for example 20 nM) and cationic lipid (e.g.,final concentration 2 μg/ml) are complexed in EGM basal media (BioWhittaker) at 37° C. for 30 minutes in polystyrene tubes. Followingvortexing, the complexed siNA is added to each well and incubated forthe times indicated. For initial optimization experiments, cells areseeded, for example, at 1×10³ in 96 well plates and siNA complex addedas described. Efficiency of delivery of siNA to cells is determinedusing a fluorescent siNA complexed with lipid. Cells in 6-well dishesare incubated with siNA for 24 hours, rinsed with PBS and fixed in 2%paraformaldehyde for 15 minutes at room temperature. Uptake of siNA isvisualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTP, 10 U RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10 U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/r×n) and normalizing to β-actin or GAPDH mRNA inparallel TAQMAN® reactions (real-time PCR monitoring of amplification).For each gene of interest an upper and lower primer and a fluorescentlylabeled probe are designed. Real time incorporation of SYBR Green I dyeinto a specific PCR product can be measured in glass capillary tubesusing a lightcyler. A standard curve is generated for each primer pairusing control cRNA. Values are represented as relative expression toGAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Models Useful to Evaluate the Down-Regulation of MyostatinGene Expression

Cell Culture

There are numerous cell culture systems that can be used to analyzereduction of myostatin levels either directly or indirectly by measuringdownstream effects. For example, cultured C2C12 cells can be used incell culture experiments to assess the efficacy of nucleic acidmolecules of the invention. As such, cells treated with nucleic acidmolecules of the invention (e.g., siNA) targeting myostatin RNA would beexpected to have decreased myostatin expression capacity compared tomatched control nucleic acid molecules having a scrambled or inactivesequence. In a non-limiting example, C2C12 cells are cultured andmyostatin expression is quantified, for example by time-resolved immunofluorometric assay. myostatin messenger-RNA expression is quantitatedwith RT-PCR. Untreated cells are compared to cells treated with siNAmolecules transfected with a suitable reagent, for example a cationiclipid such as lipofectamine, and myostatin protein and RNA levels arequantitated. Dose response assays are then performed to establish dosedependent inhibition of myostatin expression.

In several cell culture systems, cationic lipids have been shown toenhance the bioavailability of oligonucleotides to cells in culture(Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In oneembodiment, siNA molecules of the invention are complexed with cationiclipids for cell culture experiments. siNA and cationic lipid mixturesare prepared in serum-free DMEM immediately prior to addition to thecells. DMEM plus additives are warmed to room temperature (about 20-25°C.) and cationic lipid is added to the final desired concentration andthe solution is vortexed briefly. siNA molecules are added to the finaldesired concentration and the solution is again vortexed briefly andincubated for 10 minutes at room temperature. In dose responseexperiments, the RNA/lipid complex is serially diluted into DMEMfollowing the 10 minute incubation.

Animal Models

Evaluating the efficacy of myostatin agents in animal models is animportant prerequisite to human clinical trials. Lead anti-myostatinsiNA molecules chosen from in vitro assays can be further tested in thefollowing model. Bogdanovich et al., 2002, Nature, 420, 418-421, testedthe ability of inhibition of myostatin in vivo to ameliorate thedystrophic phenotype in the mdx mouse model of Duchenne musculardystrophy (DMD). The authors blocked endogenous myostatin in mdx mice byintraperitoneal injections of blocking antibodies for 3 months and foundincrease in body weight, muscle mass, muscle size, and absolute musclestrength along with a significant decrease in muscle degeneration andconcentrations of serum creatine kinase. The authors concluded thatmyostatin blockade provides a novel, pharmacologic strategy fortreatment of diseases associated with muscle wasting such as DMD, andcircumvents the major problems associated with conventional gene therapyin these disorders. Furthermore, in myostatin null mice (Mstn −/−)crossed with mdx mice, a model for Duchenne and Becker musculardystrophy, Wagner et al., 2002, Ann. Neurol. 52, 832-836, foundincreased muscle mass, increased body weight, increased muscle fibersize, and increased strength compared to Mstn +/+/mdx mice. There wasalso a reduction in the extent of muscle fibrosis in these mice. Theauthors noted that although the loss of myostatin does not correct theprimary defect in the mdx mice, it may ameliorate some features of thedystrophic phenotype.

As such, these models can be used in evaluating the efficacy of siNAmolecules of the invention in preventing muscle atrophy and wastingassociated with myopathic or dystrophic diseases and constions. Thesemodels and others can similarly be used to evaluate the safety andefficacy of siNA molecules of the invention in a pre-clinical setting.

Example 9 RNAi Mediated Inhibition of Myostatin RNA Expression

siNA constructs (Table III) are tested for efficacy in reducingmyostatin RNA expression in C2C12 cells. The siNA transfection mixturesare added to cells to give a final siNA concentration of 25 nM in avolume of 150 μl. Each siNA transfection mixture is added to 3 wells fortriplicate siNA treatments. Cells are incubated at 37° C. for 24 h inthe continued presence of the siNA transfection mixture. At 24 h, RNA isprepared from each well of treated cells. The supernatants with thetransfection mixtures are first removed and discarded, then the cellsare lysed and RNA prepared from each well. Target gene expressionfollowing treatment is evaluated by RT-PCR for the target gene and for acontrol gene (36B4, an RNA polymerase subunit) for normalization. Thetriplicate data is averaged and the standard deviations determined foreach treatment. Normalized data are graphed and the percent reduction oftarget mRNA by active siNAs in comparison to their respective invertedcontrol siNAs was determined.

Example 10 Indications

The siNA molecules of the invention can be used to prevent, inhibit,treat or prevent treat or prevent diseases, traits, or conditionsassociated with muscular atrophy, muscle weakness, muscle dysfunction,or muscle destruction, including muscular dystrophy, myotonic dystrophy,myotonia congentia, poliomyelitis, amyotrophic lateral sclerosis (ALS orLou Gehrig's disease), Guillain-Barre syndrome, muscle wasting (e.g.,age or HIV related), sarcopenia, myalgias, myopathies, hypotonis,hypotonia, cachexia, spinal cord injury, or muscle injury, or any otherrelated trait, disease or condition that is related to or will respondto the levels of myostatin in a cell or tissue, alone or in combinationwith other therapies. Nucleic acid molecules of the invention can alsobe adapted to treat or prevent obesity, diabetes (e.g., type I and typeII), and insulin resistance in a subject. Alternately the nucleic acidmolecules of the instant invention, individually, or in combination orin conjunction with other drugs, can be adapted for use to promotemuscle hypertrophy, including use for increased strength, athleticism,bodybuilding, prevention of muscle atrophy (e.g., in astronauts), orcosmetic applications in a subject or organism. In one embodiment, siNAmolecules of the invention are used in combination with anabolic orandrogenic agents as are known in the art.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with an siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I Myostatin/GDF8 Accession Numbers NM_005259 Homo sapiens growthdifferentiation factor 8 (GDF8), mRNAgi|4885258|ref|NM_005259.1|[4885258] AF104922 Homo sapiens myostatin(GDF8) mRNA, complete cds gi|4028595|gb|AF104922.1|AF104922[4028595]AC073120 Homo sapiens BAC clone RP11-612D17 from 2, complete sequencegi|15638891|gb|AC073120.5|[15638891] AF019627 Homo sapiens myostatin(MSTN) mRNA, complete cds gi|2623581|gb|AF019627.1|AF019627[2623581]AF019619 Papio hamadryas myostatin (MSTN) mRNA, complete cdsgi|2623565|gb|AF019619.1|AF019619[2623565] AY055750 Macaca fascicularismyostatin mRNA, complete cds gi|16506527|gb|AY055750.1|[16506527]AY208121 Sus scrofa myostatin gene, complete cdsgi|34484364|gb|AY208121.1|[34484364] AF097584 Equus caballus myostatin(MSTN) gene, partial cds gi|4588100|gb|AF097584.1|AF097584[4588100]AF266758 Ovis aries myostatin (MSTN) gene, exons 2 and 3 and partial cdsgi|8575725|gb|AF266758.1|AF266758[8575725] AY032689 Capra hircusmyostatin (MSTN) gene, exons 2 and 3 and partial cdsgi|13989958|gb|AY032689.1|[13989958] AJ237920 Sus scrofa MSTN gene,exons 2 and 3, partial gi|4585631|emb|AJ237920.1|SSC237920[4585631]

TABLE II Myostatin/GDF8 siNA and Target SequencesMyostatin/GDF8 NM_005259 Seq Seq Seq Pos Seq ID UPos Upper seq ID LPosLower seq ID 3 AUUCACUGGUGUGGCAAGU 1 3 AUUCACUGGUGUGGCAAGU 1 21ACUUGCCACACCAGUGAAU 158 21 UUGUCUCUCAGACUGUACA 2 21 UUGUCUCUCAGACUGUACA2 39 UGUACAGUCUGAGAGACAA 159 39 AUGCAUUAAAAUUUUGCUU 3 39AUGCAUUAAAAUUUUGCUU 3 57 AAGCAAAAUUUUAAUGCAU 160 57 UGGCAUUACUCAAAAGCAA4 57 UGGCAUUACUCAAAAGCAA 4 75 UUGCUUUUGAGUAAUGCCA 161 75AAAGAAAAGUAAAAGGAAG 5 75 AAAGAAAAGUAAAAGGAAG 5 93 CUUCCUUUUACUUUUCUUU162 93 GAAACAAGAACAAGAAAAA 6 93 GAAACAAGAACAAGAAAAA 6 111UUUUUCUUGUUCUUGUUUC 163 111 AAGAUUAUAUUGAUUUUAA 7 111AAGAUUAUAUUGAUUUUAA 7 129 UUAAAAUCAAUAUAAUCUU 164 129AAAUCAUGCAAAAACUGCA 8 129 AAAUCAUGCAAAAACUGCA 8 147 UGCAGUUUUUGCAUGAUUU165 147 AACUCUGUGUUUAUAUUUA 9 147 AACUCUGUGUUUAUAUUUA 9 165UAAAUAUAAACACAGAGUU 166 165 ACCUGUUUAUGCUGAUUGU 10 165ACCUGUUUAUGCUGAUUGU 10 183 ACAAUCAGCAUAAACAGGU 167 183UUGCUGGUCCAGUGGAUCU 11 183 UUGCUGGUCCAGUGGAUCU 11 201AGAUCCACUGGACCAGCAA 168 201 UAAAUGAGAACAGUGAGCA 12 201UAAAUGAGAACAGUGAGCA 12 219 UGCUCACUGUUCUCAUUUA 169 219AAAAAGAAAAUGUGGAAAA 13 219 AAAAAGAAAAUGUGGAAAA 13 237UUUUCCACAUUUUCUUUUU 170 237 AAGAGGGGCUGUGUAAUGC 14 237AAGAGGGGCUGUGUAAUGC 14 255 GCAUUACACAGCCCCUCUU 171 255CAUGUACUUGGAGACAAAA 15 255 CAUGUACUUGGAGACAAAA 15 273UUUUGUCUCCAAGUACAUG 172 273 ACACUAAAUCUUCAAGAAU 16 273ACACUAAAUCUUCAAGAAU 16 291 AUUCUUGAAGAUUUAGUGU 173 291UAGAAGCCAUUAAGAUACA 17 291 UAGAAGCCAUUAAGAUACA 17 309UGUAUCUUAAUGGCUUCUA 174 309 AAAUCCUCAGUAAACUUCG 18 309AAAUCCUCAGUAAACUUCG 18 327 CGAAGUUUACUGAGGAUUU 175 327GUCUGGAAACAGCUCCUAA 19 327 GUCUGGAAACAGCUCCUAA 19 345UUAGGAGCUGUUUCCAGAC 176 345 ACAUCAGCAAAGAUGUUAU 20 345ACAUCAGCAAAGAUGUUAU 20 363 AUAACAUCUUUGCUGAUGU 177 363UAAGACAACUUUUACCCAA 21 363 UAAGACAACUUUUACCCAA 21 381UUGGGUAAAAGUUGUCUUA 178 381 AAGCUCCUCCACUCCGGGA 22 381AAGCUCCUCCACUCCGGGA 22 399 UCCCGGAGUGGAGGAGCUU 179 399AACUGAUUGAUCAGUAUGA 23 399 AACUGAUUGAUCAGUAUGA 23 417UCAUACUGAUCAAUCAGUU 180 417 AUGUCCAGAGGGAUGACAG 24 417AUGUCCAGAGGGAUGACAG 24 435 CUGUCAUCCCUCUGGACAU 181 435GCAGCGAUGGCUCUUUGGA 25 435 GCAGCGAUGGCUCUUUGGA 25 453UCCAAAGAGCCAUCGCUGC 182 453 AAGAUGACGAUUAUCACGC 26 453AAGAUGACGAUUAUCACGC 26 471 GCGUGAUAAUCGUCAUCUU 183 471CUACAACGGAAACAAUCAU 27 471 CUACAACGGAAACAAUCAU 27 489AUGAUUGUUUCCGUUGUAG 184 489 UUACCAUGCCUACAGAGUC 28 489UUACCAUGCCUACAGAGUC 28 507 GACUCUGUAGGCAUGGUAA 185 507CUGAUUUUCUAAUGCAAGU 29 507 CUGAUUUUCUAAUGCAAGU 29 525ACUUGCAUUAGAAAAUCAG 186 525 UGGAUGGAAAACCCAAAUG 30 525UGGAUGGAAAACCCAAAUG 30 543 CAUUUGGGUUUUCCAUCCA 187 543GUUGCUUCUUUAAAUUUAG 31 543 GUUGCUUCUUUAAAUUUAG 31 561CUAAAUUUAAAGAAGCAAC 188 561 GCUCUAAAAUACAAUACAA 32 561GCUCUAAAAUACAAUACAA 32 579 UUGUAUUGUAUUUUAGAGC 189 579AUAAAGUAGUAAAGGCCCA 33 579 AUAAAGUAGUAAAGGCCCA 33 597UGGGCCUUUACUACUUUAU 190 597 AACUAUGGAUAUAUUUGAG 34 597AACUAUGGAUAUAUUUGAG 34 615 CUCAAAUAUAUCCAUAGUU 191 615GACCCGUCGAGACUCCUAC 35 615 GACCCGUCGAGACUCCUAC 35 633GUAGGAGUCUCGACGGGUC 192 633 CAACAGUGUUUGUGCAAAU 36 633CAACAGUGUUUGUGCAAAU 36 651 AUUUGCACAAACACUGUUG 193 651UCCUGAGACUCAUCAAACC 37 651 UCCUGAGACUCAUCAAACC 37 669GGUUUGAUGAGUCUCAGGA 194 669 CUAUGAAAGACGGUACAAG 38 669CUAUGAAAGACGGUACAAG 38 687 CUUGUACCGUCUUUCAUAG 195 687GGUAUACUGGAAUCCGAUC 39 687 GGUAUACUGGAAUCCGAUC 39 705GAUCGGAUUCCAGUAUACC 196 705 CUCUGAAACUUGACAUGAA 40 705CUCUGAAACUUGACAUGAA 40 723 UUCAUGUCAAGUUUCAGAG 197 723ACCCAGGCACUGGUAUUUG 41 723 ACCCAGGCACUGGUAUUUG 41 741CAAAUACCAGUGCCUGGGU 198 741 GGCAGAGCAUUGAUGUGAA 42 741GGCAGAGCAUUGAUGUGAA 42 759 UUCACAUCAAUGCUCUGCC 199 759AGACAGUGUUGCAAAAUUG 43 759 AGACAGUGUUGCAAAAUUG 43 777CAAUUUUGCAACACUGUCU 200 777 GGCUCAAACAACCUGAAUC 44 777GGCUCAAACAACCUGAAUC 44 795 GAUUCAGGUUGUUUGAGCC 201 795CCAACUUAGGCAUUGAAAU 45 795 CCAACUUAGGCAUUGAAAU 45 813AUUUCAAUGCCUAAGUUGG 202 813 UAAAAGCUUUAGAUGAGAA 46 813UAAAAGCUUUAGAUGAGAA 46 831 UUCUCAUCUAAAGCUUUUA 203 831AUGGUCAUGAUCUUGCUGU 47 831 AUGGUCAUGAUCUUGCUGU 47 849ACAGCAAGAUCAUGACCAU 204 849 UAACCUUCCCAGGACCAGG 48 849UAACCUUCCCAGGACCAGG 48 867 CCUGGUCCUGGGAAGGUUA 205 867GAGAAGAUGGGCUGAAUCC 49 867 GAGAAGAUGGGCUGAAUCC 49 885GGAUUCAGCCCAUCUUCUC 206 885 CGUUUUUAGAGGUCAAGGU 50 885CGUUUUUAGAGGUCAAGGU 50 903 ACCUUGACCUCUAAAAACG 207 903UAACAGACACACCAAAAAG 51 903 UAACAGACACACCAAAAAG 51 921CUUUUUGGUGUGUCUGUUA 208 921 GAUCCAGAAGGGAUUUUGG 52 921GAUCCAGAAGGGAUUUUGG 52 939 CCAAAAUCCCUUCUGGAUC 209 939GUCUUGACUGUGAUGAGCA 53 939 GUCUUGACUGUGAUGAGCA 53 957UGCUCAUCACAGUCAAGAC 210 957 ACUCAACAGAAUCACGAUG 54 957ACUCAACAGAAUCACGAUG 54 975 CAUCGUGAUUCUGUUGAGU 211 975GCUGUCGUUACCCUCUAAC 55 975 GCUGUCGUUACCCUCUAAC 55 993GUUAGAGGGUAACGACAGC 212 993 CUGUGGAUUUUGAAGCUUU 56 993CUGUGGAUUUUGAAGCUUU 56 1011 AAAGCUUCAAAAUCCACAG 213 1011UUGGAUGGGAUUGGAUUAU 57 1011 UUGGAUGGGAUUGGAUUAU 57 1029AUAAUCCAAUCCCAUCCAA 214 1029 UCGCUCCUAAAAGAUAUAA 58 1029UCGCUCCUAAAAGAUAUAA 58 1047 UUAUAUCUUUUAGGAGCGA 215 1047AGGCCAAUUACUGCUCUGG 59 1047 AGGCCAAUUACUGCUCUGG 59 1065CCAGAGCAGUAAUUGGCCU 216 1065 GAGAGUGUGAAUUUGUAUU 60 1065GAGAGUGUGAAUUUGUAUU 60 1083 AAUACAAAUUCACACUCUC 217 1083UUUUACAAAAAUAUCCUCA 61 1083 UUUUACAAAAAUAUCCUCA 61 1101UGAGGAUAUUUUUGUAAAA 218 1101 AUACUCAUCUGGUACACCA 62 1101AUACUCAUCUGGUACACCA 62 1119 UGGUGUACCAGAUGAGUAU 219 1119AAGCAAACCCCAGAGGUUC 63 1119 AAGCAAACCCCAGAGGUUC 63 1137GAACCUCUGGGGUUUGCUU 220 1137 CAGCAGGCCCUUGCUGUAC 64 1137CAGCAGGCCCUUGCUGUAC 64 1155 GUACAGCAAGGGCCUGCUG 221 1155CUCCCACAAAGAUGUCUCC 65 1155 CUCCCACAAAGAUGUCUCC 65 1173GGAGACAUCUUUGUGGGAG 222 1173 CAAUUAAUAUGCUAUAUUU 66 1173CAAUUAAUAUGCUAUAUUU 66 1191 AAAUAUAGCAUAUUAAUUG 223 1191UUAAUGGCAAAGAACAAAU 67 1191 UUAAUGGCAAAGAACAAAU 67 1209AUUUGUUCUUUGCCAUUAA 224 1209 UAAUAUAUGGGAAAAUUCC 68 1209UAAUAUAUGGGAAAAUUCC 68 1227 GGAAUUUUCCCAUAUAUUA 225 1227CAGCGAUGGUAGUAGACCG 69 1227 CAGCGAUGGUAGUAGACCG 69 1245CGGUCUACUACCAUCGCUG 226 1245 GCUGUGGGUGCUCAUGAGA 70 1245GCUGUGGGUGCUCAUGAGA 70 1263 UCUCAUGAGCACCCACAGC 227 1263AUUUAUAUUAAGCGUUCAU 71 1263 AUUUAUAUUAAGCGUUCAU 71 1281AUGAACGCUUAAUAUAAAU 228 1281 UAACUUCCUAAAACAUGGA 72 1281UAACUUCCUAAAACAUGGA 72 1299 UCCAUGUUUUAGGAAGUUA 229 1299AAGGUUUUCCCCUCAACAA 73 1299 AAGGUUUUCCCCUCAACAA 73 1317UUGUUGAGGGGAAAACCUU 230 1317 AUUUUGAAGCUGUGAAAUU 74 1317AUUUUGAAGCUGUGAAAUU 74 1335 AAUUUCACAGCUUCAAAAU 231 1335UAAGUACCACAGGCUAUAG 75 1335 UAAGUACCACAGGCUAUAG 75 1353CUAUAGCCUGUGGUACUUA 232 1353 GGCCUAGAGUAUGCUACAG 76 1353GGCCUAGAGUAUGCUACAG 76 1371 CUGUAGCAUACUCUAGGCC 233 1371GUCACUUAAGCAUAAGCUA 77 1371 GUCACUUAAGCAUAAGCUA 77 1389UAGCUUAUGCUUAAGUGAC 234 1389 ACAGUAUGUAAACUAAAAG 78 1389ACAGUAUGUAAACUAAAAG 78 1407 CUUUUAGUUUACAUACUGU 235 1407GGGGGAAUAUAUGCAAUGG 79 1407 GGGGGAAUAUAUGCAAUGG 79 1425CCAUUGCAUAUAUUCCCCC 236 1425 GUUGGCAUUUAACCAUCCA 80 1425GUUGGCAUUUAACCAUCCA 80 1443 UGGAUGGUUAAAUGCCAAC 237 1443AAACAAAUCAUACAAGAAA 81 1443 AAACAAAUCAUACAAGAAA 81 1461UUUCUUGUAUGAUUUGUUU 238 1461 AGUUUUAUGAUUUCCAGAG 82 1461AGUUUUAUGAUUUCCAGAG 82 1479 CUCUGGAAAUCAUAAAACU 239 1479GUUUUUGAGCUAGAAGGAG 83 1479 GUUUUUGAGCUAGAAGGAG 83 1497CUCCUUCUAGCUCAAAAAC 240 1497 GAUCAAAUUACAUUUAUGU 84 1497GAUCAAAUUACAUUUAUGU 84 1515 ACAUAAAUGUAAUUUGAUC 241 1515UUCCUAUAUAUUACAACAU 85 1515 UUCCUAUAUAUUACAACAU 85 1533AUGUUGUAAUAUAUAGGAA 242 1533 UCGGCGAGGAAAUGAAAGC 86 1533UCGGCGAGGAAAUGAAAGC 86 1551 GCUUUCAUUUCCUCGCCGA 243 1551CGAUUCUCCUUGAGUUCUG 87 1551 CGAUUCUCCUUGAGUUCUG 87 1569CAGAACUCAAGGAGAAUCG 244 1569 GAUGAAUUAAAGGAGUAUG 88 1569GAUGAAUUAAAGGAGUAUG 88 1587 CAUACUCCUUUAAUUCAUC 245 1587GCUUUAAAGUCUAUUUCUU 89 1587 GCUUUAAAGUCUAUUUCUU 89 1605AAGAAAUAGACUUUAAAGC 246 1605 UUAAAGUUUUGUUUAAUAU 90 1605UUAAAGUUUUGUUUAAUAU 90 1623 AUAUUAAACAAAACUUUAA 247 1623UUUACAGAAAAAUCCACAU 91 1623 UUUACAGAAAAAUCCACAU 91 1641AUGUGGAUUUUUCUGUAAA 248 1641 UACAGUAUUGGUAAAAUGC 92 1641UACAGUAUUGGUAAAAUGC 92 1659 GCAUUUUACCAAUACUGUA 249 1659CAGGAUUGUUAUAUACCAU 93 1659 CAGGAUUGUUAUAUACCAU 93 1677AUGGUAUAUAACAAUCCUG 250 1677 UCAUUCGAAUCAUCCUUAA 94 1677UCAUUCGAAUCAUCCUUAA 94 1695 UUAAGGAUGAUUCGAAUGA 251 1695AACACUUGAAUUUAUAUUG 95 1695 AACACUUGAAUUUAUAUUG 95 1713CAAUAUAAAUUCAAGUGUU 252 1713 GUAUGGUAGUAUACUUGGU 96 1713GUAUGGUAGUAUACUUGGU 96 1731 ACCAAGUAUACUACCAUAC 253 1731UAAGAUAAAAUUCCACAAA 97 1731 UAAGAUAAAAUUCCACAAA 97 1749UUUGUGGAAUUUUAUCUUA 254 1749 AAAUAGGGAUGGUGCAGCA 98 1749AAAUAGGGAUGGUGCAGCA 98 1767 UGCUGCACCAUCCCUAUUU 255 1767AUAUGCAAUUUCCAUUCCU 99 1767 AUAUGCAAUUUCCAUUCCU 99 1785AGGAAUGGAAAUUGCAUAU 256 1785 UAUUAUAAUUGACACAGUA 100 1785UAUUAUAAUUGACACAGUA 100 1803 UACUGUGUCAAUUAUAAUA 257 1803ACAUUAACAAUCCAUGCCA 101 1803 ACAUUAACAAUCCAUGCCA 101 1821UGGCAUGGAUUGUUAAUGU 258 1821 AACGGUGCUAAUACGAUAG 102 1821AACGGUGCUAAUACGAUAG 102 1839 CUAUCGUAUUAGCACCGUU 259 1839GGCUGAAUGUCUGAGGCUA 103 1839 GGCUGAAUGUCUGAGGCUA 103 1857UAGCCUCAGACAUUCAGCC 260 1857 ACCAGGUUUAUCACAUAAA 104 1857ACCAGGUUUAUCACAUAAA 104 1875 UUUAUGUGAUAAACCUGGU 261 1875AAAACAUUCAGUAAAAUAG 105 1875 AAAACAUUCAGUAAAAUAG 105 1893CUAUUUUACUGAAUGUUUU 262 1893 GUAAGUUUCUCUUUUCUUC 106 1893GUAAGUUUCUCUUUUCUUC 106 1911 GAAGAAAAGAGAAACUUAC 263 1911CAGGGGCAUUUUCCUACAC 107 1911 CAGGGGCAUUUUCCUACAC 107 1929GUGUAGGAAAAUGCCCCUG 264 1929 CCUCCAAAUGAGGAAUGGA 108 1929CCUCCAAAUGAGGAAUGGA 108 1947 UCCAUUCCUCAUUUGGAGG 265 1947AUUUUCUUUAAUGUAAGAA 109 1947 AUUUUCUUUAAUGUAAGAA 109 1965UUCUUACAUUAAAGAAAAU 266 1965 AGAAUCAUUUUUCUAGAGG 110 1965AGAAUCAUUUUUCUAGAGG 110 1983 CCUCUAGAAAAAUGAUUCU 267 1983GUUGGCUUUCAAUUCUGUA 111 1983 GUUGGCUUUCAAUUCUGUA 111 2001UACAGAAUUGAAAGCCAAC 268 2001 AGCAUACUUGGAGAAACUG 112 2001AGCAUACUUGGAGAAACUG 112 2019 CAGUUUCUCCAAGUAUGCU 269 2019GCAUUAUCUUAAAAGGCAG 113 2019 GCAUUAUCUUAAAAGGCAG 113 2037CUGCCUUUUAAGAUAAUGC 270 2037 GUCAAAUGGUGUUUGUUUU 114 2037GUCAAAUGGUGUUUGUUUU 114 2055 AAAACAAACACCAUUUGAC 271 2055UUAUCAAAAUGUCAAAAUA 115 2055 UUAUCAAAAUGUCAAAAUA 115 2073UAUUUUGACAUUUUGAUAA 272 2073 AACAUACUUGGAGAAGUAU 116 2073AACAUACUUGGAGAAGUAU 116 2091 AUACUUCUCCAAGUAUGUU 273 2091UGUAAUUUUGUCUUUGGAA 117 2091 UGUAAUUUUGUCUUUGGAA 117 2109UUCCAAAGACAAAAUUACA 274 2109 AAAUUACAACACUGCCUUU 118 2109AAAUUACAACACUGCCUUU 118 2127 AAAGGCAGUGUUGUAAUUU 275 2127UGCAACACUGCAGUUUUUA 119 2127 UGCAACACUGCAGUUUUUA 119 2145UAAAAACUGCAGUGUUGCA 276 2145 AUGGUAAAAUAAUAGAAAU 120 2145AUGGUAAAAUAAUAGAAAU 120 2163 AUUUCUAUUAUUUUACCAU 277 2163UGAUCGACUCUAUCAAUAU 121 2163 UGAUCGACUCUAUCAAUAU 121 2181AUAUUGAUAGAGUCGAUCA 278 2181 UUGUAUAAAAAGACUGAAA 122 2181UUGUAUAAAAAGACUGAAA 122 2199 UUUCAGUCUUUUUAUACAA 279 2199ACAAUGCAUUUAUAUAAUA 123 2199 ACAAUGCAUUUAUAUAAUA 123 2217UAUUAUAUAAAUGCAUUGU 280 2217 AUGUAUACAAUAUUGUUUU 124 2217AUGUAUACAAUAUUGUUUU 124 2235 AAAACAAUAUUGUAUACAU 281 2235UGUAAAUAAGUGUCUCCUU 125 2235 UGUAAAUAAGUGUCUCCUU 125 2253AAGGAGACACUUAUUUACA 282 2253 UUUUUAUUUACUUUGGUAU 126 2253UUUUUAUUUACUUUGGUAU 126 2271 AUACCAAAGUAAAUAAAAA 283 2271UAUUUUUACACUAAGGACA 127 2271 UAUUUUUACACUAAGGACA 127 2289UGUCCUUAGUGUAAAAAUA 284 2289 AUUUCAAAUUAAGUACUAA 128 2289AUUUCAAAUUAAGUACUAA 128 2307 UUAGUACUUAAUUUGAAAU 285 2307AGGCACAAAGACAUGUCAU 129 2307 AGGCACAAAGACAUGUCAU 129 2325AUGACAUGUCUUUGUGCCU 286 2325 UGCAUCACAGAAAAGCAAC 130 2325UGCAUCACAGAAAAGCAAC 130 2343 GUUGCUUUUCUGUGAUGCA 287 2343CUACUUAUAUUUCAGAGCA 131 2343 CUACUUAUAUUUCAGAGCA 131 2361UGCUCUGAAAUAUAAGUAG 288 2361 AAAUUAGCAGAUUAAAUAG 132 2361AAAUUAGCAGAUUAAAUAG 132 2379 CUAUUUAAUCUGCUAAUUU 289 2379GUGGUCUUAAAACUCCAUA 133 2379 GUGGUCUUAAAACUCCAUA 133 2397UAUGGAGUUUUAAGACCAC 290 2397 AUGUUAAUGAUUAGAUGGU 134 2397AUGUUAAUGAUUAGAUGGU 134 2415 ACCAUCUAAUCAUUAACAU 291 2415UUAUAUUACAAUCAUUUUA 135 2415 UUAUAUUACAAUCAUUUUA 135 2433UAAAAUGAUUGUAAUAUAA 292 2433 AUAUUUUUUUACAUGAUUA 136 2433AUAUUUUUUUACAUGAUUA 136 2451 UAAUCAUGUAAAAAAAUAU 293 2451AACAUUCACUUAUGGAUUC 137 2451 AACAUUCACUUAUGGAUUC 137 2469GAAUCCAUAAGUGAAUGUU 294 2469 CAUGAUGGCUGUAUAAAGU 138 2469CAUGAUGGCUGUAUAAAGU 138 2487 ACUUUAUACAGCCAUCAUG 295 2487UGAAUUUGAAAUUUCAAUG 139 2487 UGAAUUUGAAAUUUCAAUG 139 2505CAUUGAAAUUUCAAAUUCA 296 2505 GGUUUACUGUCAUUGUGUU 140 2505GGUUUACUGUCAUUGUGUU 140 2523 AACACAAUGACAGUAAACC 297 2523UUAAAUCUCAACGUUCCAU 141 2523 UUAAAUCUCAACGUUCCAU 141 2541AUGGAACGUUGAGAUUUAA 298 2541 UUAUUUUAAUACUUGCAAA 142 2541UUAUUUUAAUACUUGCAAA 142 2559 UUUGCAAGUAUUAAAAUAA 299 2559AAACAUUACUAAGUAUACC 143 2559 AAACAUUACUAAGUAUACC 143 2577GGUAUACUUAGUAAUGUUU 300 2577 CAAAAUAAUUGACUCUAUU 144 2577CAAAAUAAUUGACUCUAUU 144 2595 AAUAGAGUCAAUUAUUUUG 301 2595UAUCUGAAAUGAAGAAUAA 145 2595 UAUCUGAAAUGAAGAAUAA 145 2613UUAUUCUUCAUUUCAGAUA 302 2613 AACUGAUGCUAUCUCAACA 146 2613AACUGAUGCUAUCUCAACA 146 2631 UGUUGAGAUAGCAUCAGUU 303 2631AAUAACUGUUACUUUUAUU 147 2631 AAUAACUGUUACUUUUAUU 147 2649AAUAAAAGUAACAGUUAUU 304 2649 UUUAUAAUUUGAUAAUGAA 148 2649UUUAUAAUUUGAUAAUGAA 148 2667 UUCAUUAUCAAAUUAUAAA 305 2667AUAUAUUUCUGCAUUUAUU 149 2667 AUAUAUUUCUGCAUUUAUU 149 2685AAUAAAUGCAGAAAUAUAU 306 2685 UUACUUCUGUUUUGUAAAU 150 2685UUACUUCUGUUUUGUAAAU 150 2703 AUUUACAAAACAGAAGUAA 307 2703UUGGGAUUUUGUUAAUCAA 151 2703 UUGGGAUUUUGUUAAUCAA 151 2721UUGAUUAACAAAAUCCCAA 308 2721 AAUUUAUUGUACUAUGACU 152 2721AAUUUAUUGUACUAUGACU 152 2739 AGUCAUAGUACAAUAAAUU 309 2739UAAAUGAAAUUAUUUCUUA 153 2739 UAAAUGAAAUUAUUUCUUA 153 2757UAAGAAAUAAUUUCAUUUA 310 2757 ACAUCUAAUUUGUAGAAAC 154 2757ACAUCUAAUUUGUAGAAAC 154 2775 GUUUCUACAAAUUAGAUGU 311 2775CAGUAUAAGUUAUAUUAAA 155 2775 CAGUAUAAGUUAUAUUAAA 155 2793UUUAAUAUAACUUAUACUG 312 2793 AGUGUUUUCACAUUUUUUU 156 2793AGUGUUUUCACAUUUUUUU 156 2811 AAAAAAAUGUGAAAACACU 313 2803CAUUUUUUUGAAAGACAAA 157 2803 CAUUUUUUUGAAAGACAAA 157 2821UUUGUCUUUCAAAAAAAUG 314 The 3′-ends of the Upper sequence and the Lowersequence of the siNA construct can include an overhang sequence, forexample about 1, 2, 3, or 4 nucleotides in length, preferably 2nucleotides in length, wherein the overhanging sequence of the lowersequence is optionally complementary to a portion of the targetsequence. The upper sequence is also referred to as the sense strand,whereas the lower sequence is also referred to as the antisense strand.The upper and lower sequences in the Table can further comprise achemical modification having Formulae I-VII or any combination thereof.

TABLE III Myostalin/GDF8 Synthetic Modified siNA constructs Target SeqSeq Pos. Target ID Cmpd# Aliases Sequence ID 7 ACUGGUGUGGCAAGUUGUCUCUC315 GDF8: 9U21 sense siNA UGGUGUGGCAAGUUGUCUCTT 323 321AACUUCGUCUGGAAACAGCUCCU 316 GDF8: 323U21 sense siNACUUCGUCUGGAAACAGCUCTT 324 330 UGGAAACAGCUCCUAACAUCAGC 317GDF8: 332U21 sense siNA GAAACAGCUCCUAACAUCATT 325 522AAGUGGAUGGAAAACCCAAAUGU 318 GDF8: 524U21 sense siNAGUGGAUGGAAAACCCAAAUTT 326 871 AGAUGGGCUGAAUCCGUUUUUAG 319GDF8: 873U21 sense siNA AUGGGCUGAAUCCGUUUUUTT 327 1416UAUGCAAUGGUUGGCAUULAACC 320 GDF8: 1418U21 sense siNAUGCAAUGGUUGGCAUUUAATT 328 1425 GUUGGCAUUUAACCAUCCAAACA 321GDF8: 1427U21 sense siNA UGGCAUUUAACCAUCCAAATT 329 1926ACACCUCCAAAUGAGGAAUGGAU 322 GDF8: 1928U21 sense siNAACCUCCAAAUGAGGAAUGGTT 330 7 ACUGGUGUGGCAAGUUGUCUCUC 315GDF8: 27L21 Antisense siNA (9C) GAGACAACUUGCCACACCATT 331 321AACUUCGUCUGGAAACAGCUCCU 316 GDF8: 341L21 antisense siNAGAGCUGUUUCCAGACGAAGTT 332 (323C) 330 UGGAAACAGCUCCUAACAUCAGC 317GDF8: 350L21 antisense siNA UGAUGUUAGGAGCUGUUUCTT 333 (332C) 522AAGUGGAUGGAAAACCCAAAUGU 318 GDF8: 542L21 antisense siNAAUUUGGGUUUUCCAUCCACTT 334 (524C) 871 AGAUGGGCUGAAUCCGUUUUUAG 319GDF8: 891L21 antisense siNA AAAAACGGAUUCAGCCCAUTT 335 (873C) 1416UAUGCAAUGGUUGGCAUUUAACC 320 GDF8: 1436L21 antisense siNAUUAAAUGCCAACCAUUGCATT 336 (1418C) 1425 GUUGGCAUUUAACCAUCCAAACA 321GDF8: 1445L21 antisense siNA UUUGGAUGGUUAAAUGCCATT 337 (1427C) 1926ACACCUCCAAAUGAGGAAUGGAU 322 GDF8: 1946L21 antisense siNACCAUUCCUCAUUUGGAGGUTT 338 (1928C) 7 ACUGGUGUGGCAAGUUGUCUCUC 315GDF8: 9U21 sense siNA stab04 B uGGuGuGGcAAGuuGucucTT B 339 321AACUUCGUCUGGAAACAGCUCCU 316 GDF8: 323U21 sense siNA stab04B cuucGucuGGAAAcAGcucTT B 340 330 UGGAAACAGCUCCUAACAUCAGC 317GDF8: 323U21 sense siNA stab04 B GAAAcAGcuccuAAcAucATT B 341 522AAGUGGAUGGAAAACCCAAAUGU 318 GDF8: 524U21 sense siNA stab04B GuGGAuGGAAAAcccAAAuTT B 342 871 AGAUGGGCUGAAUCCGUUUUUAG 319GDF8: 873U21 sense siNA stab04 B AuGGGcuGAAuccGuuuuuTT B 343 1416UAUGCAAUGGUUGGCAUUUAACC 320 GDF8: 1418U21 sense siNA stab04B uGcAAuGGuuGGcAuuuAATT B 344 1425 GUUGGCAUUUAACCAUCCAAACA 321GDF8: 1427U21 sense siNA stab04 B uGGcAuuuAAccAuccAAATT B 345 1926ACACCUCCAAAUGAGGAAUGGAU 322 GDF8: 1928U21 sense siNA stab04B AccuccAAAuGAGGAAuGGTT B 346 7 ACUGGUGUGGCAAGUUGUCUCUC 315GDF8: 27L21 antisense siNA (9C) GAGAcAAcuuGccAcAccATsT 347 stab05 321AACUUCGUCUGGAAACAGCUCCU 316 GDF8: 341L21 antisense siNAGAGcuGuuuccAGAcGAAGTsT 348 (323C) stab05 330 UGGAAACAGCUCCUAACAUCAGC 317GDF8: 350L21 antisense siNA uGAuGuuAGGAGcuGuuucTsT 349 (332C) stab05 522AAGUGGAUGGAAAACCCAAAUGU 318 GDF8: 542L21 antisense siNAAuuuGGGuuuuccAuccAcTsT 350 (524C) stab05 871 AGAUGGGCUGAAUCCGUUUUUAG 319GDF8: 891L21 antisense siNA AAAAAcGGAuucAGcccAuTsT 351 (873C) stab051416 UAUGCAAUGGUUGGCAUUUAACC 320 GDF8: 1436L21 antisense siNAuuAAAuGccAAccAuuGcATsT 352 (1418C) stab05 1425 GUUGGCAUUUAACCAUCCAAACA321 GDF8: 1445L21 antisense siNA uuuGGAuGGuuAAAuGccATsT 353(1427C) stab05 1926 ACACCUCCAAAUGAGGAAUGGAU 322GDF8: 1946L21 antisense siNA ccAuuccucAuuuGGAGGuTsT 354 (1928C) stab05 7ACUGGUGUGGCAAGUUGUCUCUC 315 GDF8: 9U21 sense siNA stab07B uGGuGuGGcAAGuuGucucTT B 355 321 AACUUCGUCUGGAAACAGCUCCU 316GDF8: 323U21 sense siNA stab07 B cuucGucuGGAAAcAGcucTT B 356 330UGGAAACAGCUCCUAACAUCAGC 317 GDF8: 332U21 sense siNA stab07B GAAAcAGcuccuAAcAucATT B 357 522 AAGUGGAUGGAAAACCCAAAUGU 318GDF8: 524U21 sense siNA stab07 B GuGGAuGGAAAAcccAAAuTT B 358 871AGAUGGGCUGAAUCCGUUUUUAG 319 GDF8: 873U21 sense siNA stab07B AuGGGcuGAAuccGuuuuuTT B 359 1416 UAUGCAAUGGUUGGCAUUUAACC 320GDF8: 1418U21 sense siNA stab07 B uGcAAuGGuuGGcAuuuAATT B 360 1425GUUGGCAUUUAACCAUCCAAACA 321 GDF8: 1427U21 sense siNA stab07B uGGcAuuuAAccAuccAAATT B 361 1926 ACACCUCCAAAUGAGGAAUGGAU 322GDF8: 1928U21 sense siNA stab07 B AccuccAAAuGAGGAAuGGTT B 362 7ACUGGUGUGGCAAGUUGUCUCUC 315 GDF8: 27L21 antisense siNA (9C)GAGAcAAcuuGccAcAccATsT 363 stab11 321 AACUUCGUCUGGAAACAGCUCCU 316GDF8: 341L21 antisense siNA GAGcuGuuuccAGAcGAAGTsT 364 (323C) stab11 330UGGAAACAGCUCCUAACAUCAGC 317 GDF8: 350L21 antisense siNAuGAuGuuAGGAGcuGuuucTsT 365 (332C) stab11 522 AAGUGGAUGGAAAACCCAAAUGU 318GDF8: 542L21 antisense siNA AuuuGGGuuuuccAuccAcTsT 366 (524C) stab11 871AGAUGGGCUGAAUCCGUUUUUAG 319 GDF8: 891L21 antisense siNAAAAAAcGGAuucAGcccAuTsT 367 (873C) stab11 1416 UAUGCAAUGGUUGGCAUUUAACC320 GDF8: 1436L21 antisense siNA uuAAAuGccAAccAuuGcATsT 368(1418C) stab11 1425 GUUGGCAUUUAACCAUCCAAACA 321GDF8: 1445L21 antisense siNA uuuGGAuGGuuAAAuGccATsT 369 (1427C) stab111926 ACACCUCCAAAUGAGGAAUGGAU 322 GDF8: 1946L21 antisense siNAccAuuccucAuuuGGAGGuTsT 370 (1928C) stab11 7 ACUGGUGUGGCAAGUUGUCUCUC 315GDF8: 9U21 sense siNA stab18 B uGGuGuGGcAAGuuGucucTT B 371 321AACUUCGUCUGGAAACAGCUCCU 316 GDF8: 323U21 sense siNA stab18B cuucGucuGGAAAcAGcucTT B 372 330 UGGAAACAGCUCCUAACAUCAGC 317GDF8: 332U21 sense siNA stab18 B GAAAcAGcuccuAAcAucATT B 373 522AAGUGGAUGGAAAACCCAAAUGU 318 GDF8: 524U21 sense siNA stab18B GuGGAuGGAAAAcccAAAuTT B 374 871 AGAUGGGCUGAAUCCGUUUUUAG 319GDF8: 873U21 sense siNA stab18 B AuGGGcuGAAuccGuuuuuTT B 375 1416UAUGCAAUGGUUGGCAUUUAACC 320 GDF8: 1418U21 sense siNA stab18B uGcAAuGGuuGGcAuuuAATT B 376 1425 GUUGGCAUUUAACCAUCCAAACA 321GDF8: 1427U21 sense siNA Stab18 B uGGcAuuuAAccAuccAAATT B 377 1926ACACCUCCAAAUGAGGAAUGGAU 322 GDF8: 1928U21 sense siNA stab18B AccuccAAAuGAGGAAuGGTT B 378 7 ACUGGUGUGGCAAGUUGUCUCUC 315GDF8: 27L21 antisense siNA (9C) GAGAcAAcuuGccAcAccATsT 379 stab08 321AACUUCGUCUGGAAACAGCUCCU 316 GDF8: 341L21 antisense siNAGAGcuGuuuccAGAcGAAGTsT 380 (323C) stab08 330 UGGAAACAGCUCCUAACAUCAGG 317GDF8: 350L21 antisense siNA uGAuGuuAGGAGcuGuuucTsT 381 (332C) stab08 522AAGUGGAUGGAAAACCCAAAUGU 318 GDF8: 542L21 antisensa siNAAuuuGGGuuuuccAuccAcTsT 382 (524C) stab08 871 AGAUGGGCUGAAUCCGUUUUUAG 319GDF8: 891L21 antisense siNA AAAAAcGGAuucAGcccAuTsT 383 (873C) stab081416 UAUGCAAUGGUUGGCAUUUAACC 320 GDF8: 1436L21 antisense siNAuuAAAuGccAAccAuuGcATsT 384 (1418C) stab08 1425 GUUGGCAUUUAACCAUCCAAACA321 GDF8: 1445L21 antisense siNA uuuGGAuGGuuAAAuGccATsT 385(1427C) stab08 1926 ACACCUCCAAAUGAGGAAUGGAU 322GDF8: 1946L21 antisense siNA ccAuuccucAuuuGGAGGuTsT 386 (1928C) stab08 7ACUGGUGUGGCAAGUUGUCUCUC 315 37277 GDF8: 9U21 sense siNA stab09B UGGUGUGGCAAGUUGUCUCTT B 387 321 AACUUCGUCUGGAAACAGCUCCU 316 37278GDF8: 323U21 sense siNA stab09 B CUUCGUCUGGAAACAGCUCTT B 388 330UGGAAACAGCUCCUAACAUCAGC 317 37279 GDF8: 332U21 sense siNA Stab09B GAAACAGCUCCUAACAUCATT B 389 522 AAGUGGAUGGAAAACCCAAAUGU 318 37280GDF8: 524U21 sense siNA stab09 B GUGGAUGGAAAACCCAAAUTT B 390 871AGAUGGGCUGAAUCCGUUUUUAG 319 37281 GDF8: 873U21 sense siNA stab09B AUGGGCUGAAUCCGUUUUUTT B 391 1416 UAUGCAAUGGUUGGCAUUUAACC 320 37282GDF8: 1418U21 sense siNA stab09 B UGCAAUGGUUGGCAUUUAATT B 392 1425GUUGGCAUUUAACCAUCCAAACA 321 37283 GDF8: 1427U21 sense siNA stab09B UGGCAUUUAACCAUCCAAATT B 393 1926 ACACCUCCAAAUGAGGAAUGGAU 322 37284GDF8: 1928U21 sense siNA stab09 B ACCUCCAAAUGAGGAAUGGTT B 394 7ACUGGUGUGGCAAGUUGUCUCUC 315 GDF8: 27L21 antisense siNA (9C)GAGACAACUUGCCACACCATsT 395 stab10 321 AACUUCGUCUGGAAACAGCUCCU 316GDF8: 341L21 antisense siNA GAGCUGUUUCCAGACGAAGTsT 396 (323C) stab10 330UGGAAACAGCUCCUAACAUCAGC 317 GDF8: 350L21 antisense siNAUGAUGUUAGGAGCUGUUUCTsT 397 (332C) stab10 522 AAGUGGAUGGAAAACCCAAAUGU 318GDF8: 542L21 antisense siNA AUUUGGGUUUUCCAUCCACTsT 398 (524C) stab10 871AGAUGGGCUGAAUCCGUUUUUAG 319 GDF8: 891L21 antisense siNAAAAAACGGAUUCAGCCCAUTsT 399 (873C) stab10 1416 UAUGCAAUGGUUGGCAUUUAACC320 GDF8: 1436L21 antisense siNA UUAAAUGCCAACCAUUGCATsT 400(1418C) stab10 1425 GUUGGCAUUUAACCAUCCAAACA 321GDF8: 1445L21 antisense siNA UUUGGAUGGUUAAAUGCCATsT 401 (1427C) stab101926 ACACCUCCAAAUGAGGAAUGGAU 322 GDF8: 1946L21 antisense siNACCAUUCCUCAUUUGGAGGUTsT 402 (1928C) stab10 7 ACUGGUGUGGCAAGUUGUCUCUC 315GDF8: 27L21 antisense siNA (9C) GAGAcAAcuuGccAcAccATT B 403 stab19 321AACUUCGUCUGGAAACAGCUCCU 316 GDF8: 341L21 antisense siNAGAGcuGuuuccAGAcGAAGTT B 404 (323C) stab19 330 UGGAAACAGCUCCUAACAUCAGC317 GDF8: 350L21 antisense siNA uGAuGuuAGGAGcuGuuucTT B 405(332C) stab19 522 AAGUGGAUGGAAAACCCAAAUGU 318GDF8: 542L21 antisense siNA AuuuGGGuuuuccAuccAcTT B 406 (524C) stab19871 AGAUGGGCUGAAUCCGUJUUUAG 319 GDF8: 891L21 antisense siNAAAAAAcGGAuucAGcccAuTT B 407 (873C) stab19 1416 UAUGCAAUGGUUGGCAUUUAACC320 GDF8: 1436L21 antisense siNA uuAAAuGccAAccAuuGcATT B 408(1418C) stab19 1425 GUUGGCAUUUAACCAUCCAAACA 321GDF8: 1445L21 antisense siNA uuuGGAuGGuuAAAuGccATT B 409 (1427C) stab191926 ACACCUCCAAAUGAGGAAUGGAU 322 GDF8: 1946L21 antisense siNAccAuuccucAuuuGGAGGuTT B 410 (1928C) stab19 7 ACUGGUGUGGCAAGUUGUCUCUC 31537285 GDF8: 27L21 antisense siNA (9C) GAGACAACUUGCCACACCATT B 411 stab22321 AACUUCGUCUGGAAACAGCUCCU 316 37286 GDF8: 341L21 antisense siNAGAGCUGUUUCCAGACGAAGTT B 412 (323C) stab22 330 UGGAAACAGCUCCUAACAUCAGC317 37287 GDF8: 350L21 antisense siNA UGAUGUUAGGAGCUGUUUCTT B 413(332C) stab22 522 AAGUGGAUGGAAAACCCAAAUGU 318 37288GDF8: 542L21 antisense siNA AUUUGGGUUUUCCAUCCACTT B 414 (524C) stab22871 AGAUGGGCUGAAUCCGUUUUUAG 319 37289 GDF8: 891L21 antisense siNAAAAAACGGAUUCAGCCCAUTT B 415 (873C) stab22 1416 UAUGCAAUGGUUGGCAUUUAACC320 37290 GDF8: 1436L21 antisense siNA UUAAAUGCCAACCAUUGCATT B 416(1418C) stab22 1425 GUUGGCAUUUAACCAUCCAAACA 321 37291GDF8: 1445L21 antisense siNA UUUGGAUGGUUAAAUGCCATT B 417 (1427C) stab221926 ACACCUCCAAAUGAGGAAUGGAU 322 37292 GDF8: 1946L21 antisense siNACCAUUCCUCAUUUGGAGGUTT B 418 (1928C) stab22 Uppercase = ribonucleotide T= thymidine s = phosphorothioate linkage G = deoxy Guanosine G =2′-0-methyl Guanosine u, c = 2′-deoxy-2′-fluoro U, C B = inverted deoxyabasic A = deoxy Adenosine A = 2′-0-methyl Adenosine

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at 3′-ends S/AS “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All linkages Usually AS“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and 3′-ends — Usually S “Stab 5” 2′-fluoro Ribo — 1 at3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and 3′-ends — Usually S“Stab 7” 2′-fluoro 2′-deoxy 5′ and 3′-ends — Usually S “Stab 8”2′-fluoro 2′-O-Methyl — 1 at 3′-end Usually AS “Stab 9” Ribo Ribo 5′ and3′-ends — Usually S “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab11” 2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA5′ and 3′-ends Usually S “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS“Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 16” Ribo2′-O-Methyl 5′ and 3′-ends Usually S “Stab 17” 2′-O-Methyl 2′-O-Methyl5′ and 3′-ends Usually S “Stab 18” 2′-fluoro 2′-O-Methyl 5′ and 3′-endsUsually S “Stab 19” 2′-fluoro 2′-O-Methyl 3′-end Usually AS “Stab 20”2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-endUsually AS “Stab 22” Ribo Ribo 3′-end- Usually AS “Stab 23” 2′-fluoro*2′-deoxy* 5′ and 3′-ends Usually S “Stab 24” 2′-fluoro* 2′-O-Methyl* — 1at 3′-end Usually AS “Stab 25” 2′-fluoro* 2′-O-Methyl* — 1 at 3′-endUsually AS CAP = any terminal cap, see for example FIG. 10. All Stab00-25 chemistries can comprise 3′-terminal thymidine (TT) residues AllStab 00-25 chemistries typically comprise about 21 nucleotides, but canvary as described herein. S = sense strand AS = antisense strand *Stab23 has single ribonucleotide adjacent to 3′-CAP *Stab 24 has singleribonucleotide at 5′-terminus *Stab 25 has three ribonucleotides at5′-terminus

TABLE V A. 2.5 μmol Synthesis Cycle ABI 394 Instrument ReagentEquivalents Amount Wait Time* DNA Wait Time* 2′-O-methyl Wait Time*RNAPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl Imidazole 186 233 μL 5 sec 5 sec 5 sec TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Reagent Equivalents AmountWait Time* DNA Wait Time* 2′-O-methyl Wait Time*RNA Phosphoramidites 1531 μL 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min465 sec Acetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl Imidazole1245 124 μL 5 sec 5 sec 5 sec TCA 700 732 μL 10 sec 10 sec 10 sec Iodine20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Time* 2′-O- Reagent2′-O-methyl/Ribo methyl/Ribo Wait Time* DNA methyl Wait Time* RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-EthylTetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-MethylImidazole 502/502/502 50/50/50 μL 10 sec 10 sec 10 sec TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

1. A chemically modified short interfering nucleic acid (siNA) molecule,wherein: (a) the siNA molecule comprises a sense strand and a separateantisense strand, each strand having one or more pyrimidine nucleotidesand one or more purine nucleotides; (b) each strand is independently 18to 27 nucleotides in length, and together comprise a duplex havingbetween 17 and 23 base pairs; (c) the antisense strand is complementaryto a human myostatin RNA sequence comprising SEQ ID NO:441; (d) aplurality of the pyrimidine nucleotides present in the sense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and a plurality of the purinenucleotides present in the sense strand are 2′-deoxy purine nucleotides;and (e) a plurality of the pyrimidine nucleotides in the antisensestrand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and a plurality ofthe purine nucleotides present in the antisense strand are 2′-O-methylpurine nucleotides.
 2. The siNA molecule of claim 1, wherein the sensestrand has a cap at both 5′- and 3′-ends of the sense strand.
 3. ThesiNA molecule of claim 1 comprising a 3′-overhang on one or bothstrands.
 4. The siNA molecule of claim 1, wherein the antisense strandhas a phosphorothioate internucleotide linkage at the 3′-end.
 5. Acomposition comprising the siNA molecule of claim 1 and apharmaceutically acceptable carrier or diluent.